Dimerizing agents, their production and use

ABSTRACT

Materials and methods are disclosed for regulation of biological events such as target gene transcription and growth, proliferation or differentiation of engineered cells.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. 119(e)to copending U.S. Provisional Application No. 60/074,584, includingAppendix A, filed Feb. 13, 1998, the entire contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Rapamycin is a macrolide antibiotic produced by Streptomyceshygroscopicus which binds to a FK506-binding protein, FKBP, with highaffinity to form a rapamycin:FKBP complex. Reported Kd values for thatinteraction are as low as 200 pM. The rapamycin:FKBP complex binds withhigh affinity to the large cellular protein, FRAP, to form a tripartite,[FKBP:rapamycin]:[FRAP], complex. In that complex rapamycin acts as adimerizer or adapter to join FKBP to FRAP.

A number of naturally occurring FK506 binding proteins (FKBPs) areknown. See e.g. Kay, 1996, Biochem. J. 314:361–385 (review).FKBP-derived domains have been incorporated in the design of chimericproteins for use in biological switches in genetically engineered cells.Such switches rely upon ligand-mediated multimerization of the proteincomponents to trigger a desired biological event. See e.g. Spencer etal, 1993, Science 262:1019–1024 and PCT/US94/01617. While the potentimmunosuppressive activity of FK506 would limit its utility as amultimerizing agent, especially in animals, dimers of FK506 (and relatedcompounds) can be made which lack such immunosuppressive activity. Suchdimers have been shown to be effective for multimerizing chimericproteins containing FKBP-derived ligand binding domains.

Rapamycin, like FK506, is also capable of multimerizing appropriatelydesigned chimeric proteins. Biological switches using rapamycin orvarious derivatives or analogs thereof (“rapalogs”) as multimerizingagents have been disclosed (see WO96/41865). In the case of rapamycinitself, its significant biological activities, including potentimmunosuppressive activity, rather severely limit its use in biologicalswitches in certain applications, especially those in animals or animalcells which are sensitive to rapamycin. Improved rapalogs for suchapplications, especially rapalogs with reduced immunosuppressiveactivity, would be very desirable.

A large number of structural variants of rapamycin have been reported,typically arising as alternative fermentation products or from syntheticefforts to improve the compound's therapeutic index as animmunosuppressive agent. For example, the extensive literature onanalogs, homologs, derivatives and other compounds related structurallyto rapamycin include, among others, variants of rapamycin having one ormore of the following modifications relative to rapamycin:demethylation, elimination or replacement of the methoxy at C7, C42and/or C29; elimination, derivatization or replacement of the hydroxy atC13, C43 and/or C28; reduction, elimination or derivatization of theketone at C14, C24 and/or C30; replacement of the 6-membered pipecolatering with a 5-membered prolyl ring; and alternative substitution on thecyclohexyl ring or replacement of the cyclohexyl ring with a substitutedcyclopentyl ring. Additional historical information is presented in thebackground sections of U.S. Pat. Nos. 5,525,610; 5,310,903 and5,362,718.

U.S. Pat. No. 5,527,907 is illustrative of the patent literature. Thatdocument discloses a series of compounds which were synthesized in aneffort to make immunosuppressive rapalogs with reduced side effects. Thecompounds are disclosed via seven generic structural formulas, eachfollowed by extensive lists (two to five or more columns of text each)setting forth possible substituents at various positions on therapamycin ring. The document includes over 180 synthetic examples. Themany structural variants of that invention were reported to be potentimmunosuppressive agents.

SUMMARY OF THE INVENTION

A New Class of Rapalogs

As noted above, several decades of rather focused and competitiveresearch in the laboratories of some of the leading pharmaceutical firmsand academic institutions have created a rich literature describing awide variety of rapalogs. As reported in that literature, each portionof the rapamycin molecule which was considered susceptible to chemicalmodification was in turn modified, and the resultant rapalogs evaluatedfor utility, usually as immunosuppressive agents.

It was against that extensive background that we made the completelyunexpected discovery that led to the present invention. Briefly, whileattempting to repeat the previously described replacement of the C7methoxy group of rapamycin with an allyl or methallyl moiety and recoverthe desired C7-substituted rapalogs, we instead recovered theunintentionally synthesized members of a previously unknown family ofrapalogs and found those compounds to possess useful and in fact quitesignificant biochemical properties. Reagents and conditions used forintroducing the substitution at C3 may be used not just with rapamycinas the starting material, but also with a wide variety of rapalogs whichcan be obtained as alternative fermentation products or by syntheticmodification of rapamycin. Moreover, the C3-substituted rapalogs maythemselves be subjected to a wide variety of subsequent chemicalmodifications such as have been reported in the case of rapamycin. Thus,the discovery of our methodology for C3 substitution provides access toan entirely new and diverse family of rapalogs.

Our first novel rapalogs of this series are 3-allyl-rapamycin (R═H) and3-methallyl-rapamycin (R=methyl):

These compounds are particularly noteworthy for their ability to form atripartite complex with FKBP12 and a genetically engineered FRB domain,or with fusion proteins containing those FKBP and FRB domains. Thisbinding property and its significance is discussed in detail below. Asmentioned above, the substitution at C3 may be combined with one or moreother modifications to the rapamycin structure, numerous examples ofwhich are known in the art. Among others, these include C3 rapalogs with(a) replacement moieties at any one or more of positions 13, 14, 20, 24,28, and 30; (b) a prolyl moiety in place of the otherwise characteristicpipicolate moiety; and/or (c) variations on or in place of thesubstituted cyclohexyl ring above the pipicolate moiety as the structureis drawn above. These are disclosed in greater detail below.

These new rapalogs (“C3 rapalogs”) may thus be defined as compoundswhich comprise the structure of Formula I:

bearing one or more optional substituents, and typically unsaturated atone or more carbon—carbon bonds spanning carbons 1 through 8, as asubstantially pure stereoisomer or mixture of stereoisomers, or apharmaceutically acceptable derivative thereof (as that term is definedbelow), where R^(C3) is other than H. For example, in variousembodiments of the invention, R^(C3) is a substituted or unsubstitutedaliphatic, heteroaliphatic, aromatic or heteroaromatic moiety.

C3 rapalogs useful in practicing this invention may contain substituentsin any of the possible stereoisomeric orientations, and may comprise onestereoisomer substantially free of other stereoisomers (>90%, andpreferably >95%, free from other stereoisomers on a molar basis) or maycomprise a mixture of stereoisomers.

One preferred class of such compounds have a substantially reducedimmunosuppressive effect as compared with rapamycin. By a “substantiallyreduced immunosuppressive effect” we mean that the rapalog has less than0.1, preferably less than 0.01, and even more preferably, less than0.005 times the immunosuppressive effect observed or expected with anequimolar amount of rapamycin, as measured either clinically or in anappropriate in vitro or in vivo surrogate of human immunosuppressiveactivity, preferably carried out on tissues of lymphoid origin, oralternatively, that the rapalog yields an EC50 value in such an in vitroassay which is at least ten times, preferably at least 100 times andmore preferably at least 250 times larger than the EC50 value observedfor rapamycin in the same assay.

One appropriate in vitro surrogate of immunosuppression in a humanpatient is inhibition of human T cell proliferation in vitro. This is aconventional assay approach that may be conducted in a number of wellknown variations using various human T cells or cells lines, includingamong others human PBLs and Jurkat cells. A rapalog may thus be assayedfor human immunosuppressive activity and compared with rapamycin. Adecrease in immunosuppressive activity relative to rapamycin measured inan appropriate in vitro assay is predictive of a decrease inimmunosuppressive activity in humans, relative to rapamycin. Such invitro assays may be used to evaluate the rapalog's relativeimmunosuppressive activity.

A variety of illustrative examples of such rapalogs are disclosedherein. This class of C3 rapalogs includes, among others, those whichbind to human FKBP12, or inhibit its rotamase activity, within an orderof magnitude of results obtained with rapamycin in any conventional FKBPbinding or rotamase assay. One class of C3 rapalogs which is ofparticular interest (and is exemplified by our first two C3 rapalogs)are those compounds which comprise the structure of formula I in whichR^(C3) comprises:

bearing one or more optional substituents, whether as a substantiallypure stereoisomer or mixture of stereoisomers, or a pharmaceuticallyacceptable derivative thereof, where R¹, R⁴, R⁵, R⁶ and R⁷ are each H ora substituted or unsubstituted aliphatic, aromatic, heteroaliphatic orheteroaromatic moiety. In some embodiments the C3 substituent maycomprise a cyclic moiety, e.g., where R¹ and R⁴, R⁴ and R⁵, or R⁵ and R⁶are covalently linked together to form a ring.

Another class of C3 rapalogs of particular interest are those compoundsof Formula II:

wherein R^(C3) is other than H, and

-   R^(C30) is halo, —OR³ or (═O);-   R^(c24) is ═O, ═NR⁴═NOR⁴, ═NNHR⁴, —NHOR⁴, —NHNHR⁴, —OR⁴, —OC(O)R⁴,    —OC(O)NR⁴, halo or —H;-   R^(C13) and R^(C28) are independently H, halo, —OR³, —OR⁵, —OC(O)R⁵,    —OC(O)NHR⁵, —SR⁵, —SC(O)R⁵, —SC(O)NHR⁵, —NR⁵R^(5′) or    —N(R⁵)(CO)R^(5′);-   R^(C14) is ═O, —OR⁶, —NR⁶, —H, —NC(O)R⁶, —OC(O)R⁶ or —OC(O)NR⁶;-   R³ is H, —R⁷, —C(O)R⁷ or —C(O)NHR⁷ or a cyclic moiety (e.g.,    carbonate) bridging C28 and C30; and,-   R^(C29) is H or OR¹¹ (e.g., OH or OMe);    where each substituent may be present in either stereochemical    orientation unless otherwise indicated, and where each occurrence of    R¹, R⁴, R⁵R⁶, R⁷, R⁹, R¹⁰ and R¹¹ is independently selected from H,    aliphatic, heteroaliphatic, aryl and heteroaryl; and R⁸ is H, halo,    —CN, ═O, —OH, —NR⁹R¹⁰, OSO₂CF₃, OSO₂F, OSO₂R⁴′, OCOR^(4′),    OCONR^(4′) R^(5′), or OCON(OR^(4′))R^(5′),    as a substantially pure stereoisomer or mixture of stereoisomers, or    a pharmaceutically acceptable derivative thereof.

Another class of C3 rapalogs of special interest includes those in whichone or both of R^(C13) and R^(C28) is are independently H, halo, —OR³,—OR⁵, —OC(O)R⁵, OC(O)NHR⁵, —SR⁵, —SC(O)R⁵—SC(O)NHR⁵, —NR⁵R^(5′) or—N(R⁵)(CO)R^(5′) where each halo moiety is independently selected fromF, Cl, Br and I.

Another class of C3 rapalogs of special interest are those in which oneor both of R^(C24) and R^(C30) are other than ═O. This class includes24, 30-tetrahydro-C3 substituted rapamycins and mono and diethersthereof and the 24-halo, 30-halo and 24,30-dihalo derivatives thereof.

Another class of C3 rapalogs which are of particular interest arerapalogs of Formula I wherein n is 1. This class of rapalogs includesrapalogs comprising a prolyl ring system in place of a pipicolate ringsystem.

Another class of C3 rapalogs which are of particular interest arerapalogs of Formula II wherein moiety “a” is other than

This class of rapalogs include the class of 43-epi-rapalogs in which thehydroxyl moiety at position 43 has the opposite stereochemicalorientation with that shown immediately above, is a mixture ofstereoisomers of the 43-hydroxyl group or contains derivatives of any ofthe foregoing, including ethers, esters, carbamates, halides and otherderivatives of any of the foregoing position 43 rapalogs. This classfurther includes rapalogs in which the cyclohexyl ring is otherwisesubstituted and/or contains 5 ring atoms in place of the characteristicsubstituted cyclohexyl ring of rapamycin.

Subsets of the foregoing classes of C3 rapalogs further differ instructure from rapamycin with respect to one or more additionalstructural features (e.g. one or both substituents at C7, for instance),as set forth above in connection with Formula II or in connection withany of the other classes of C3 rapalogs noted herein.

Methods for Producing C3 Rapalogs

This invention further provides methods for producing C3 rapalogs whichinvolve subjecting rapamycin or a rapalog starting material to reagentsand conditions permitting replacement of the C3 hydrogen with the desiremoiety. The product may be recovered from the reaction mixture, andpurified from unreacted starting materials and side products. Theproduct may be subjected to one or more additional transformations priorto final purification. Methods and materials for C3 substitution andproduct recovery are disclosed in detail below.

Methods for Multimerizing Chimeric Proteins

This invention further provides methods and materials for multimerizingchimeric proteins in genetically engineered cells using the new rapalogsdisclosed herein, preferably while avoiding the immunosuppressiveeffects of rapamycin.

The genetically engineered cells contain one or more recombinant nucleicacid constructs encoding specialized chimeric proteins as describedherein. Typically a first chimeric protein contains one or more FKBPdomains which are capable of binding to a C3 rapalog of this invention.This first chimeric protein is also referred to herein as an “FKBPfusion protein” and further comprises at least one protein domainheterologous to at least one of its FKBP domains. The complex formed bythe binding of the FKBP fusion protein to the C3 rapalog is capable ofbinding to a second chimeric protein which contains one or more FRBdomains (the “FRB fusion protein”). The FRB fusion protein furthercomprises at least one protein domain heterologous to at least one ofits FRB domains. In some embodiments, the FKBP fusion protein and theFRB fusion protein are different from one another. In other embodiments,however, the FKBP fusion protein is also an FRB fusion protein. In thoseembodiments, the chimeric protein comprises one or more FKBP domains aswell as one or more FRB domains. In such cases, the first and secondchimeric proteins may be the same protein, may be referred to asFKBP-FRB fusion proteins and contain at least one domain heterologous tothe FKBP and/or FRB domains.

The chimeric proteins may be readily designed, based on incorporation ofappropriately chosen heterologous domains, such that theirmultimerization triggers one or more of a wide variety of desiredbiological responses. The nature of the biological response triggered byrapalog-mediated complexation is determined by the choice ofheterologous domains in the fusion proteins. The heterologous domainsare therefore referred to as “action” or “effector” domains. Thegenetically engineered cells for use in practicing this invention willcontain one or more recombinant nucleic acid constructs encoding thechimeric proteins, and in certain applications, will further contain oneor more accessory nucleic acid constructs, such as one or more targetgene constructs. Illustrative biological responses, applications of thesystem and types of accessory nucleic acid constructs are discussed indetail below.

A system involving related materials and methods is disclosed in WO96/41865 (Clackson et al) and is expected to be useful in a variety ofapplications including, among others, research uses and therapeuticapplications. That system involves the use of a multimerizing agentcomprising rapamycin or a rapalog of the generic formula:

wherein U is —H, —OR¹, —SR¹, —OC(O)R¹, —OC(O)NHR¹, —NHR¹, —NHC(O)R¹,NHSO₂—R¹ or —R²; R² is a substituted aryl or allyl or alkylaryl (e.g.benzyl or substituted benzyl); V is —OR³ or (═O); W is ═O, ═NR⁴ ═NOR⁴,═NNHR⁴, —NHOR⁴, —NHNHR⁴, —OR⁴, —OC(O)R⁴, —OC(O)NR⁴ or —H; Y is —OR⁵,—OC(O)R⁵ or —OC(O)NHR⁵; Z is ═O, —OR⁶, —NR⁶, —H, —NC(O)R⁶, —OC(O)R⁶ or—OC(O)NR⁶; R³ is H, —R⁷, —C(O)R⁷, —C(O)NHR⁷ or C-28/C-30 cycliccarbonate; and R⁴ is H or alkyl; where R¹, R⁴, R⁵, R⁶ and R⁷ areindependently selected from H, alkyl, alkylaryl or aryl, as those termsare defined in WO 96/41865. A number of rapalogs are specificallydisclosed in that document.

The subject invention is based upon a system similar to that disclosedin WO 96/41865, but involves the use of C3 rapalogs as the multimerizingagents. The subject invention thus provides a method for multimerizingchimeric proteins in cells which comprises (a) providing appropriatelyengineered cells containing nucleic acid constructs for directing theexpression of the desired chimeric protein(s) and any desired accessoryrecombinant constructs, and (b) contacting the cells with a C3 rapalogor a pharmaceutically acceptable derivative thereof as described herein.The rapalog forms a complex containing itself and at least two moleculesof the chimeric protein(s).

In one embodiment, at least one of the chimeric proteins contains atleast one FKBP domain whose peptide sequence differs from a naturallyoccurring FKBP peptide sequence, e.g. the peptide sequence of humanFKBP12, at up to ten amino acid residues in the peptide sequence.Preferably the number of changes in peptide sequence is limited to five,and more preferably to 1, 2, or 3. Preferably the changes arereplacements rather than simple deletions or insertions. In embodimentsin which the rapalog differs from rapamycin at one or more positions inaddition to the modification at C3, loss of C7 methoxy and loss oftriene conjugation, at least one of the chimeric proteins may contain atleast one FKBP domain comprising at least one amino acid replacementrelative to the sequence of a naturally occurring FKBP, especially amammalian FKBP such as human FKBP12.

In another embodiment, at least one of the chimeric proteins contains atleast one FRB domain whose peptide sequence differs from a naturallyoccurring FRB peptide sequence, e.g. the FRB domain of human FRAP, at upto ten amino acid residues in the peptide sequence. Preferably thenumber of changes in peptide sequence is limited to five, and morepreferably to 1, 2, or 3. Mutations of particular interest includereplacement of one or more of T2098, D2102, Y2038, F2039, K2095 of anFRB domain derived from human FRAP with independently selectedreplacement amino acids, e.g., A, N, H, L, or S. Also of interest arethe replacement of one or more of F1975, F1976, D2039 and N2035 of anFRB domain derived from yeast TOR1, or the replacement of one or more ofF1978, F1979, D2042 and N2038 of an FRB domain derived from yeast TOR2,with independently selected replacement amino acids, e.g. H, L, S, A orV.

In certain embodiments the chimeric protein(s) contain at least onemodification in peptide sequence, preferably up to three modifications,relative to naturally occurring sequences, in both one or more FKBPdomains and one or more FRB domains.

As mentioned previously, in the various embodiments of this invention,the chimeric protein(s) contain one or more “action” or “effector”domains which are heterologous with respect to the FKBP and/or FRBdomains. Effector domains may be selected from a wide variety of proteindomains including DNA binding domains, transcription activation domains,cellular localization domains and signaling domains (i.e., domains whichare capable upon clustering or multimerization, of triggering cellgrowth, proliferation, differentiation, apoptosis, gene transcription,etc.). A variety of illustrative effector domains which may be used inpracticing this invention are disclosed in the various scientific andpatent documents cited herein.

For example, in certain embodiments, one fusion protein contains atleast one DNA binding domain (e.g., a GAL4 or ZFHD1 DNA-binding domain)and another fusion protein contains at least one transcriptionactivation domain (e.g., a VP16 or p65 transcription activation domain).Ligand-mediated association of the fusion proteins represents theformation of a transcription factor complex and leads to initiation oftranscription of a target gene linked to a DNA sequence recognized by(i.e., capable of binding with) the DNA-binding domain on one of thefusion proteins.

In other embodiments, one fusion protein contains at least one domaincapable of directing the fusion protein to a particular cellularlocation such as the cell membrane, nucleus, ER or other organelle orcellular component. Localization domains which target the cell membrane,for example, include domains such as a myristoylation site or atransmembrane region of a receptor protein or other membrane-spanningprotein. Another fusion protein can contain a signaling domain capable,upon membrane localization and/or clustering, of activating a cellularsignal transduction pathway. Examples of signaling domains include anintracellular domain of a growth factor or cytokine receptor, anapoptosis triggering domain such as the intracellular domain of FAS orTNF-R1, and domains derived from other intracellular signaling proteinssuch as SOS, Raf, 1ck, ZAP-70, etc. A number of signaling proteins aredisclosed in PCT/US94/01617 (see e.g. pages 23–26). In still otherembodiments, each of the fusion proteins contains at least one FRBdomain and at least one FKBP domain, as well as one or more heterologousdomains. Such fusion proteins are capable of homodimerization andtriggering signaling in the presence of the rapalog. In general, domainscontaining peptide sequence endogenous to the host cell are preferred inapplications involving whole organisms. Thus, for human gene therapyapplications, domains of human origin are of particular interest.

Recombinant nucleic acid constructs encoding the fusion proteins arealso provided, as are nucleic acid constructs capable of directing theirexpression, and vectors containing such constructs for introducing theminto cells, particularly eukaryotic cells, of which yeast and animalcells are of particular interest. In view of the constituent componentsof the fusion proteins, the recombinant DNA molecules which encode themare capable of selectively hybridizing (a) to a DNA molecule encoding apolypeptide comprising an FRB domain or FKBP domain and (b) to a DNAmolecule encoding the heterologous domain or a protein from which theheterologous protein domain was derived. DNAs are also encompassed whichwould be capable of so hybridizing but for the degeneracy of the geneticcode.

Using nucleic acid sequences encoding the fusion proteins, nucleic acidconstructs for directing their expression in eukaryotic cells, andvectors or other means for introducing such constructs into cells,especially animal cells, one may genetically engineer cells,particularly animal cells, preferably mammalian cells, and mostpreferably human cells, for a number of important uses. To do so, onefirst provides an expression vector or nucleic acid construct fordirecting the expression in a eukaryotic (preferably animal) cell of thedesired chimeric protein(s) and then introduces the recombinant DNA intothe cells in a manner permitting DNA uptake and expression of theintroduced DNA in at least a portion of the cells. One may use any ofthe various methods and materials for introducing DNA into cells forheterologous gene expression, a variety of which are well known and/orcommercially available.

One object of this invention is thus a method for multimerizing fusionproteins, such as described herein, in cells, preferably animal cells.To recap, one of the fusion proteins is capable of binding to a C3rapalog of this invention and contains at least one FKBP domain and atleast one domain heterologous thereto. The second fusion proteincontains at least one FRB domain and at least one domain heterologousthereto and is capable of forming a tripartite complex with the firstfusion protein and one or more molecules of the C3 rapalog. In someembodiments one or more of the heterologous domains present on one ofthe fusion proteins are also present on the other fusion protein, i.e.,the two fusion proteins have one or more common heterologous domains. Inother embodiments, each fusion protein contains one or more differentheterologous domains.

The method comprises contacting appropriately engineered cells with theC3 rapalog by adding the rapalog to the culture medium in which thecells are located or administering the rapalog to the organism in whichthe cells are located. The cells are preferably eukaryotic cells, morepreferably animal cells, and most preferably mammalian cells. Primatecells, especially human cells, are of particular interest.Administration of the C3 rapalog to a human or non-human animal may beeffected using any pharmaceutically acceptable formulation and route ofadministration. Oral administration of a pharmaceutically acceptablecomposition containing the C3 rapalog together with one or morepharmaceutically acceptable carriers, buffers or other excipients iscurrently of greatest interest.

A specific object of this invention is a method, as otherwise describedabove, for inducing transcription of a target gene in arapalog-dependent manner. The cells typically contain, in addition torecombinant DNAs encoding the two fusion proteins, a target geneconstruct which comprises a target gene operably linked to a DNAsequence which is responsive to the presence of a complex of the fusionproteins with rapamycin or a rapalog. The target gene construct may berecombinant, and the target gene and/or a regulatory nucleic acidsequence linked thereto may be heterologous with respect to the hostcell. In certain embodiments the cells are responsive to contact with aC3 rapalog which binds to the FKBP fusion protein and participates in acomplex with a FRB fusion protein with a detectable preference overbinding to endogenous FKBP and/or FRB-containing proteins of the hostcell.

Another specific object of this invention is a method, as otherwisedescribed above, for inducing cell death in a rapalog-dependent manner.In such cells, at least one of the heterologous domains on at least onefusion protein, and usually two fusion proteins, is a domain such as theintracellular domain of FAS or TNF-R1, which, upon clustering, triggersapoptosis of the cell.

Another specific object of this invention is a method, as otherwisedescribed above, for inducing cell growth, differentiation orproliferation in a rapalog-dependent manner. In such cells, at least oneof the heterologous domains of at least one of the fusion proteins is asignaling domain such as, for example, the intracellular domain of areceptor for a hormone which mediates cell growth, differentiation orproliferation, or a downstream mediator of such signaling. Cell growth,differentiation and/or proliferation follows clustering of suchsignaling domains. Such clustering occurs in nature following hormonebinding, and in engineered cells of this invention following contactwith a C3 rapalog.

Cells of human origin are preferred for human gene therapy applications,although cell types of various origins (human or other species) may beused, and may, if desired, be encapsulated within a biocompatiblematerial for use in human subjects.

Also provided are materials and methods for producing the foregoingengineered cells. This object is met by providing recombinant nucleicacids, typically DNA molecules, encoding the fusion proteins, togetherwith any desired ancillary recombinant nucleic acids such as a targetgene construct, and introducing the recombinant nucleic acids into thehost cells under conditions permitting nucleic acid uptake by cells.Such transfection may be effected ex vivo, using host cells maintainedin culture. Cells that are engineered in culture may subsequently beintroduced into a host organism, e.g. in ex vivo gene therapyapplications. Doing so thus constitutes a method for providing a hostorganism, preferably a human or non-human mammal, which is responsive(as described herein) to the presence of a C3 rapalog as providedherein. Alternatively transfection may be effected in vivo, using hostcells present in a human or non-human host organism. In such cases, thenucleic acid molecules are introduced directly into the host organismunder conditions permitting uptake of nucleic acids by one or more ofthe host organism's cells. This approach thus constitutes an alternativemethod for providing a host organism, preferably a human or non-humanmammal, which is responsive (as described herein) to the presence of aC3 rapalog. Various materials and methods for the introduction of DNAand RNA into cells in culture or in whole organisms are known in the artand may be adapted for use in practicing this invention.

Other objects are achieved using the engineered cells described herein.For instance, a method is provided for multimerizing fusion proteins ofthis invention by contacting cells engineered as described herein withan effective amount of the C3 rapalog permitting the rapalog to form acomplex with the fusion proteins. In embodiments in whichmultimerization of the fusion proteins triggers transcription of atarget gene, this constitutes a method for activating the expression ofthe target gene. In embodiments in which the fusion proteins contain oneor more signaling domains, this constitutes a method for activating acellular signal transduction pathway. In specific embodiments in whichthe signaling domains are selected based on their ability followingclustering to trigger cell growth, proliferation, differentiation orcell death, C3 rapalog-mediated clustering constitutes a method foractuating cell growth, proliferation, differentiation or cell death, asthe case may be. These methods may be carried out in cell culture or inwhole organisms, including human patients. In the former case, therapamycin or rapalog is added to the culture medium. In the latter case,the rapamycin or rapalog (which may be in the form of a pharmaceuticalor veterinary composition) is administered to the whole organism, e.g.,orally, parenterally, etc. Preferably, the dose of the C3 rapalogadministered to an animal is below the dosage level that would causeundue immunosuppression in the recipient.

Also disclosed are kits for use in the genetic engineering of cells orhuman or non-human animals as described herein. One such kit containsone or more recombinant nucleic acid constructs encoding fusion proteinsof this invention. The recombinant nucleic acid constructs willgenerally be in the form of eukaryotic expression vectors suitable forintroduction into animal cells and capable of directing the expressionof the fusion proteins therein. Such vectors may be viral vectors asdescribed elsewhere herein. The kit may also contain a sample of a C3rapalog of this invention capable of forming a complex with the encodedfusion proteins. The kit may further contain a multimerizationantagonist such as FK506 or some other compound capable of binding toone of the fusion proteins but incapable of forming a complex with both.In certain embodiments, the recombinant nucleic acid constructs encodingthe fusion proteins will contain a cloning site in place of DNA encodingone or more of the heterologous domains, thus permitting thepractitioner to introduce DNA encoding a heterologous domain of choice.In some embodiments the kit may also contain a target gene constructcontaining a target gene or cloning site linked to a DNA sequenceresponsive to the presence of the complexed fusion proteins, asdescribed in more detail elsewhere. The kit may contain a package insertidentifying the enclosed nucleic acid construct(s), and/or instructionsfor introducing the construct(s) into host cells or organisms.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The definitions and orienting information below will be helpful for afull understanding of this document.

FRB domains are polypeptide regions (protein “domains”), typically of atleast about 89 amino acid residues, which are capable of forming atripartite complex with an FKBP protein and rapamycin (or a C3 rapalogof this invention). FRB domains are present in a number of naturallyoccurring proteins, including FRAP proteins (also referred to in theliterature as “RAPT1” or RAFT”) from human and other species; yeastproteins including Tor1 and Tor2; and a Candida FRAP homolog.Information concerning the nucleotide sequences, cloning, and otheraspects of these proteins is already known in the art, permitting thesynthesis or cloning of DNA encoding the desired FRB peptide sequence,e.g., using well known methods and PCR primers based on publishedsequences.

protein source reference/sequence accession numbers human FRAP Brown etal, 1994, Nature 369, 756–758; GenBank accession #L34075, NCBI Seq ID508481; Chiu et al, 1994, PNAS USA 91, 12574–12578; Chen et al, 1995,PNAS USA 92, 4947–4951 murine Chiu et al, supra. RAPT1 yeast Tor1Helliwell et al, 1994, Mol Cell Biol 5, 105–118; EMBL Accession #X74857,NCBI Seq Id #468738 yeast Tor2 Kunz et al, 1993, Cell 73, 585–596; EMBLAccession #X71416, NCBI Seq ID 298027 Candida TOR WO95/33052 (Berlin etal)

FRB domains for use in this invention generally contain at least about89–100 amino acid residues. FIG. 2 of Chiu et al, supra, displays a160-amino acid span of human FRAP, murine FRAP, S. cerevisiae TOR1 andS. cerevisiae TOR2 encompassing the conserved FRB region. Typically theFRB sequence selected for use in fusion proteins of this invention willspan at least the 89-amino acid sequence Glu-39 through Lys/Arg-127, asthe sequence is numbered in that figure. For reference, using thenumbering of Chen et al or Sabitini et al, the 89-amino acid sequence isnumbered Glu-2025 through Lys-2113 in the case of human FRAP, Glu-1965through Lys-2053 in the case of Tor2, and Glu-1962 through Arg-2050 inthe case of Tor1. An FRB domain for use in fusion proteins of thisinvention will be capable of binding to a complex of an FKBP proteinbound to rapamycin or a C3 rapalog of this invention (as may bedetermined by any means, direct or indirect, for detecting such binding,including, for example, means for detecting such binding employed in theFRAP/RAFT/RAPT and Tor-related references cited herein). The peptidesequence of such an FRB domain comprises (a) a naturally occurringpeptide sequence spanning at least the indicated 89-amino acid region ofthe proteins noted above or corresponding regions of homologousproteins; (b) a variant of a naturally occurring FRB sequence in whichup to about ten (preferably 1–5, more preferably 1–3) amino acids of thenaturally-occurring peptide sequence have been deleted, inserted, orreplaced with substitute amino acids; or (c) a peptide sequence encodedby a DNA sequence capable of selectively hybridizing to a DNA moleculeencoding a naturally occurring FRB domain or by a DNA sequence whichwould be capable, but for the degeneracy of the genetic code, ofselectively hybridizing to a DNA molecule encoding a naturally occurringFRB domain. A preferred FRB triple mutant is disclosed in theExperimental Examples below.

FKBPs (FK506 binding proteins) are the cytosolic receptors formacrolides such as FK506, FK520 and rapamycin and are highly conservedacross species lines. For the purpose of this disclosure, FKBPs areproteins or protein domains which are capable of binding to rapamycin orto a C3 rapalog of this invention and further forming a tripartitecomplex with an FRB-containing protein. An FKBP domain may also bereferred to as a “rapamycin binding domain”. Information concerning thenucleotide sequences, cloning, and other aspects of various FKBP speciesis already known in the art, permitting the synthesis or cloning of DNAencoding the desired FKBP peptide sequence, e.g., using well knownmethods and PCR primers based on published sequences. See e.g. Staendartet al, 1990, Nature 346, 671–674 (human FKBP12); Kay, 1996, Biochem. J.314, 361–385 (review). Homologous FKBP proteins in other mammalianspecies, in yeast, and in other organisms are also known in the art andmay be used in the fusion proteins disclosed herein. See e.g. Kay, 1996,Biochem. J. 314, 361–385 (review). The size of FKBP domains for use inthis invention varies, depending on which FKBP protein is employed. AnFKBP domain of a fusion protein of this invention will be capable ofbinding to rapamycin or a C3 rapalog of this invention and participatingin a tripartite complex with an FRB-containing protein (as may bedetermined by any means, direct or indirect, for detecting suchbinding). The peptide sequence of an FKBP domain of an FKBP fusionprotein of this invention comprises (a) a naturally occurring FKBPpeptide sequence, preferably derived from the human FKBP12 protein(exemplified below) or a peptide sequence derived from another humanFKBP, from a murine or other mammalian FKBP, or from some other animal,yeast or fungal FKBP; (b) a variant of a naturally occurring FKBPsequence in which up to about ten (preferably 1–5, more preferably 1–3,and in some embodiments just one) amino acids of the naturally-occurringpeptide sequence have been deleted, inserted, or replaced withsubstitute amino acids; or (c) a peptide sequence encoded by a DNAsequence capable of selectively hybridizing to a DNA molecule encoding anaturally occurring FKBP or by a DNA sequence which would be capable,but for the degeneracy of the genetic code, of selectively hybridizingto a DNA molecule encoding a naturally occurring FKBP.

“Capable of selectively hybridizing” as that phrase is used herein meansthat two DNA molecules are susceptible to hybridization with oneanother, despite the presence of other DNA molecules, underhybridization conditions which can be chosen or readily determinedempirically by the practitioner of ordinary skill in this art. Suchtreatments include conditions of high stringency such as washingextensively with buffers containing 0.2 to 6×SSC, and/or containing 0.1%to 1% SDS, at temperatures ranging from room temperature to 65–750 C.See for example F. M. Ausubel et al., Eds, Short Protocols in MolecularBiology, Units 6.3 and 6.4 (John Wiley and Sons, New York, 3d Edition,1995).

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein.

“Nucleic acid constructs”, as that term is used herein, denote nucleicacids (usually DNA, but also encompassing RNA, e.g. in a retroviraldelivery system) used in the practice of this invention which aregenerally recombinant, as that term is defined below, and which mayexist in free form (i.e., not covalently linked to other nucleic acidsequence) or may be present within a larger molecule such as a DNAvector, retroviral or other viral vector or a chromosome of agenetically engineered host cell. Nucleic acid constructs of particularinterest are those which encode fusion proteins of this invention orwhich comprise a target gene and expression control elements. Theconstruct may further include nucleic acid portions comprising one ormore of the following elements relevant to regulation of transcription,translation, and/or other processing of the coding region or geneproduct thereof: transcriptional promoter and/or enhancer sequences, aribosome binding site, introns, etc.

“Recombinant”, “chimeric” and “fusion”, as those terms are used herein,denote materials comprising various component domains, sequences orother components which are mutually heterologous in the sense that theydo not occur together in the same arrangement, in nature. Morespecifically, the component portions are not found in the samecontinuous polypeptide or nucleotide sequence or molecule in nature, atleast not in the same cells or order or orientation or with the samespacing present in the chimeric protein or recombinant DNA molecule ofthis invention.

“Transcription control element” denotes a regulatory DNA sequence, suchas initiation signals, enhancers, and promoters, which induce or controltranscription of protein coding sequences with which they are operablylinked. The term “enhancer” is intended to include regulatory elementscapable of increasing, stimulating, or enhancing transcription from apromoter. Such transcription regulatory components can be presentupstream of a coding region, or in certain cases (e.g. enhancers), inother locations as well, such as in introns, exons, coding regions, and3′ flanking sequences.

“Dimerization”, “oligomerization” and “multimerization” are usedinterchangeably herein and refer to the association or clustering of twoor more protein molecules, mediated by the binding of a drug to at leastone of the proteins. In preferred embodiments, the multimerization ismediated by the binding of two or more such protein molecules to acommon divalent or multivalent drug. The formation of a complexcomprising two or more protein molecules, each of which containing oneor more FKBP domains, together with one or more molecules of an FKBPligand which is at least divalent (e.g. FK1012 or AP1510) is an exampleof such association or clustering. In cases where at least one of theproteins contains more than one drug binding domain, e.g., where atleast one of the proteins contains three FKBP domains, the presence of adivalent drug leads to the clustering of more than two proteinmolecules. Embodiments in which the drug is more than divalent (e.g.trivalent) in its ability to bind to proteins bearing drug bindingdomains also can result in clustering of more than two proteinmolecules. The formation of a tripartite complex comprising a proteincontaining at least one FRB domain, a protein containing at least oneFKBP domain and a molecule of rapamycin is another example of suchprotein clustering. In certain embodiments of this invention, fusionproteins contain multiple FRB and/or FKBP domains. Complexes of suchproteins may contain more than one molecule of rapamycin or a derivativethereof or other dimerizing agent and more than one copy of one or moreof the constituent proteins. Again, such multimeric complexes are stillreferred to herein as tripartite complexes to indicate the presence ofthe three types of constituent molecules, even if one or more arerepresented by multiple copies. The formation of complexes containing atleast one divalent drug and at least two protein molecules, each ofwhich contains at least one drug binding domain, may be referred to as“oligomerization” or “multimerization”, or simply as “dimerization”,“clustering” or association”.

“Dimerizer” denotes a C3 rapalog of this invention which brings togethertwo or more proteins in a multimeric complex.

“Activate” as applied herein to the expression or transcription of agene denotes a directly or indirectly observable increase in theproduction of a gene product.

“Genetically engineered cells” denotes cells which have been modified(“transduced”) by the introduction of recombinant or heterologousnucleic acids (e.g. one or more DNA constructs or their RNAcounterparts) and further includes the progeny of such cells whichretain part or all of such genetic modification.

A “therapeutically effective dose” of a C3 rapalog of this inventiondenotes a treatment- or prophylaxis-effective dose, e.g., a dose whichyields detectable target gene transcription or cell growth,proliferation, differentiation, death, etc. in the geneticallyengineered cell, or a dose which is predicted to be treatment- orprophylaxis-effective by extrapolation from data obtained in animal orcell culture models. A therapeutically effective dose is usuallypreferred for the treatment of a human or non-human mammal.

This invention involves methods and materials for multimerizing chimericproteins in genetically engineered cells using C3 rapalogs. The designand implementation of various dimerization-based biological switches hasbeen reported, inter alia, in Spencer et al and in various internationalpatent applications cited herein. Other accounts of successfulapplication of this general approach have also been reported. Chimericproteins containing an FRB domain fused to an effector domain has alsobeen disclosed in Rivera et al, 1996, Nature Medicine 2, 1028–1032 andin WO 96/41865 (Clackson et al) and WO 95/33052 (Berlin et al). As notedpreviously, the fusion proteins are designed such that association ofthe effector domains, through ligand-mediated “dimerization” or“multimerization” of the fusion proteins which contain them, triggers adesired biological event such as transcription of a desired gene, celldeath, cell proliferation, etc. For example, clustering of chimericproteins containing an action domain derived from the intracellularportion of the T cell receptor CD3 zeta domain triggers transcription ofa gene under the transcriptional control of the IL-2 promoter orpromoter elements derived therefrom. In other embodiments, the actiondomain comprises a domain derived from the intracellular portion of aprotein such as FAS or the TNFalpha receptor (TNFalpha-R1), which arecapable, upon oligomerization, of triggering apoptosis of the cell. Instill other embodiments, the action domains comprise a DNA-bindingdomain such as GAL4 or ZFHD1 and a transcription activation domain suchas VP16 or p65, paired such that oligomerization of the chimericproteins represents assembly of a transcription factor complex whichtriggers transcription of a gene linked to a DNA sequence recognized by(capable of specific binding interaction with) the DNA binding domain.

Chimeric proteins containing one or more ligand-binding domains and oneor more action domains, e.g. for activation of transcription of a targetgene, triggering cell death or other signal transduction pathway,cellular localization, etc., are disclosed in PCT/US94/01617,PCT/US94/08008 and Spencer et al, supra. The design and use of suchchimeric proteins for ligand-mediated gene-knock out and forligand-mediated blockade of gene expression or inhibition of geneproduct function are disclosed in PCT/US95/10591. Novel DNA bindingdomains and DNA sequences to which they bind which are useful inembodiments involving regulated transcription of a target gene aredisclosed, e.g., in Pomeranz et al, 1995, Science 267:93–96. Thosereferences provide substantial information, guidance and examplesrelating to the design, construction and use of DNA constructs encodinganalogous chimeras, target gene constructs, and other aspects which mayalso be useful to the practitioner of the subject invention.

By appropriate choice of chimeric proteins, this invention permits oneto activate the transcription of a desired gene; actuate cell growth,proliferation, differentiation or apoptosis; or trigger other biologicalevents in engineered cells in a rapalog-dependent manner analogous tothe systems described in the patent documents and other references citedabove. The engineered cells, preferably animal cells, may be growing ormaintained in culture or may be present within whole organisms, as inthe case of human gene therapy, transgenic animals, and other suchapplications. The rapalog is administered to the cell culture or to theorganism containing the engineered cells, as the case may be, in anamount effective to multimerize the FKBP fusion proteins and FRB fusionproteins (as may be observed indirectly by monitoring target genetranscription, apoptosis or other biological process so triggered). Inthe case of administration to whole organisms, the rapalog may beadministered in a composition containing the rapalog and one or moreacceptable veterinary or pharmaceutical diluents and/or excipients.

A compound which binds to one of the chimeric proteins but does not formtripartite complexes with both chimeric proteins may be used as amultimerization antagonist. As such it may be administered to theengineered cells, or to organisms containing them (preferably in acomposition as described above in the case of administration to wholeanimals), in an amount effective for blocking or reversing the effect ofthe rapalog, i.e. for preventing, inhibiting or disruptingmultimerization of the chimeras. For instance, FK506, FK520 or any ofthe many synthetic FKBP ligands which do not form tripartite complexeswith FKBP and FRAP may be used as an antagonist.

One important aspect of this invention provides materials and methodsfor rapalog-dependent, direct activation of transcription of a desiredgene. In one such embodiment, a set of two or more different chimericproteins, and corresponding DNA constructs capable of directing theirexpression, is provided. One such chimeric protein contains as itsaction domain(s) one or more transcriptional activation domains. Theother chimeric protein contains as its action domain(s) one or moreDNA-binding domains. A rapalog of this invention is capable of bindingto both chimeras to form a dimeric or multimeric complex thus containingat least one DNA binding domain and at least one transcriptionalactivating domain. Formation of such complexes leads to activation oftranscription of a target gene linked to, and under the transcriptionalcontrol of, a DNA sequence to which the DNA-binding domain is capable ofbinding, as can be observed by monitoring directly or indirectly thepresence or concentration of the target gene product.

Preferably the DNA binding domain, and a chimera containing it, binds toits recognized DNA sequence with sufficient selectivity so that bindingto the selected DNA sequence can be observed (directly or indirectly)despite the presence of other, often numerous other, DNA sequences.Preferably, binding of the chimera comprising the DNA-binding domain tothe selected DNA sequence is at least two, more preferably three andeven more preferably more than four orders of magnitude greater thanbinding to any one alternative DNA sequence, as measured by in vitrobinding studies or by measuring relative rates or levels oftranscription of genes associated with the selected DNA sequence ascompared with any alternative DNA sequences.

Cells which have been genetically engineered to contain such a set ofconstructs, together with any desired accessory constructs, may be usedin applications involving ligand-mediated, regulated actuation of thedesired biological event, be it regulated transcription of a desiredgene, regulated triggering of a signal transduction pathway such as thetriggering of apoptosis, or another event. Cells engineered forregulatable expression of a target gene, for instance, can be used forregulated production of a desired protein (or other gene product)encoded by the target gene. Such cells may be grown in culture byconventional means. Addition of the rapalog to the culture mediumcontaining the cells leads to expression of the target gene by the cellsand production of the protein encoded by that gene. Expression of thegene and production of the protein can be turned off by withholdingfurther multimerization agent from the media, by removing residualmultimerization agent from the media, or by adding to the medium amultimerization antagonist reagent.

Engineered cells of this invention can also be produced and/or used invivo, to modify whole organisms, preferably animals, especially humans,e.g. such that the cells produce a desired protein or other resultwithin the animal containing them. Such uses include gene therapyapplications.

Embodiments involving regulatable actuation of apoptosis provideengineered cells susceptible to rapalog-inducible cell death. Suchengineered cells can be eliminated from a cell culture or host organismafter they have served their intended purposed (e.g. production of adesired protein or other product), if they have or develop unwantedproperties, or if they are no longer useful, safe or desired.Elimination is effected by adding the rapalog to the medium oradministering it to the host organism. In such cases, the action domainsof the chimeras are protein domains such as the intracellular domains ofFAS or TNF-R1, downstream components of their signaling pathways orother protein domains which upon oligomerization trigger apoptosis.

This invention thus provides materials and methods for achieving abiological effect in cells in response to the addition of a rapalog ofthis invention. The method involves providing cells engineered asdescribed herein and exposing the cells to the rapalog.

For example, this invention provides a method for activatingtranscription of a target gene in cells. The method involves providingcells containing (a) DNA constructs encoding a set of chimeric proteinsof this invention capable upon rapalog-mediated multimerization ofinitiating transcription of a target gene and (b) a target gene linkedto an associated cognate DNA sequence responsive to the multimerizationevent (e.g. a DNA sequence recognized, i.e., capable of binding with, aDNA-binding domain of a foregoing chimeric protein. The method involvesexposing the cells to a rapalog capable of binding to the chimericproteins in an amount effective to result in expression of the targetgene. In cases in which the cells are growing in culture, exposing thecells to the rapalog may be effected by adding the rapalog to theculture medium. In cases in which the cells are present within a hostorganism, exposing them to the rapalog is effected by administering therapalog to the host organism. For instance, in cases in which the hostorganism is a human or non-human, the rapalog may be administered to thehost organism by oral, bucal, sublingual, transdermal, subcutaneous,intramuscular, intravenous, intra-joint or inhalation administration inan appropriate vehicle therefor. Again, depending on the design of theconstructs for the chimeric proteins and of any accessory constructs,the rapalog-mediated biological event may be activation of a cellularfunction such as signal transduction leading to cell growth, cellproliferation, gene transcription, or apoptosis; deletion of a gene ofinterest, blockade of expression of a gene of interest, or inhibition offunction of a gene product of interest; direct transcription of a geneof interest; etc.

This invention further encompasses a pharmaceutical compositioncomprising a rapalog of this invention in admixture with apharmaceutically acceptable carrier and optionally with one or morepharmaceutically acceptable excipients. Such pharmaceutical compositionscan be used to promote multimerization of chimeras of this invention inengineered cells in whole animals, e.g. in human gene therapyapplications to achieve any of the objectives disclosed herein.

Said differently, this invention provides a method for achieving any ofthose objectives, e.g. activation of transcription of a target gene(typically a heterologous gene for a therapeutic protein), cell growthor proliferation, cell death or some other selected biological event, inan animal, preferably a human patient, in need thereof and containingengineered cells of this invention. That method involves administeringto the animal a pharmaceutical composition containing the rapalog by aroute of administration and in an amount effective to causemultimerization of the chimeric proteins in at least a portion of theengineered cells. Multimerization may be detected indirectly bydetecting the occurrence of target gene expression; cell growth,proliferation or death; or other objective for which the chimeras weredesigned and the cells genetically engineered.

This invention further encompasses a pharmaceutical compositioncomprising a multimerization antagonist of this invention in admixturewith a pharmaceutically acceptable carrier and optionally with one ormore pharmaceutically acceptable excipients for inhibiting or otherwisereducing, in whole or part, the extent of multimerization of chimericproteins in engineered cells of this invention in a subject, and thusfor de-activating the transcription of a target gene, for example, orturning off another biological result of this invention. Thus, the useof the multimerizing rapalogs and of the multimerization antagonistreagents to prepare pharmaceutical compositions and achieve theirpharmacologic results is encompassed by this invention.

Also disclosed is a method for providing a host organism, preferably ananimal, typically a non-human mammal or a human subject, responsive to arapalog of this invention. The method involves introducing into theorganism cells which have been engineered in accordance with thisinvention, i.e. containing one or more nucleic acid constructs encodingthe chimeric proteins, and so forth. The engineered cells may beencapsulated using any of a variety of materials and methods beforebeing introduced into the host organism. Alternatively, one canintroduce the nucleic acid constructs of this invention into a hostorganism, e.g. a mammal, under conditions permitting incorporationthereof into one or more cells of the host mammal, e.g. using viralvectors, introduction of DNA by injection or via catheter, etc.

Also provided are kits for producing cells responsive to a rapalog ofthis invention. One such kit contains one or more nucleic acidconstructs encoding and capable of directing the expression of chimeraswhich, upon rapalog-mediated oligomerization, trigger the desiredbiological response. The kit may contain a quantity of a rapalog capableof multimerizing the chimeric protein molecules encoded by theconstruct(s) of the kit, and may contain in addition a quantity of amultimerization antagonist. The kit may further contain a nucleic acidconstruct encoding a target gene (or cloning site) linked to a cognateDNA sequence which is recognized by the dimerized chimeric proteinspermitting transcription of a gene linked to that cognate DNA sequencein the presence of multimerized chimeric protein molecules. Theconstructs may be associated with one or more selection markers forconvenient selection of transfectants, as well as other conventionalvector elements useful for replication in prokaryotes, for expression ineukaryotes, and the like. The selection markers may be the same ordifferent for each different construct, permitting the selection ofcells which contain each such construct(s).

The accessory construct for introducing into cells a target gene inassociation with a cognate DNA sequence may contain a cloning site inplace of a target gene. A kit containing such a construct permits theengineering of cells for regulatable expression of a gene to be providedby the practitioner.

Other kits of this invention may contain one or two (or more) nucleicacid constructs for chimeric proteins in which one or more contain acloning site in place of the transcriptional activator or DNA bindingprotein, permitting the user to insert whichever such domain s/hewishes. Such a kit may optionally include other elements as describedabove, e.g. a nucleic construct for a target gene with or without acognate DNA sequence for a pre-selected DNA binding domain.

Any of the kits may also contain positive control cells which werestably transformed with constructs of this invention such that theyexpress a reporter gene (for CAT, SEAP, beta-galactosidase or anyconveniently detectable gene product) in response to exposure of thecells to the rapalog. Reagents for detecting and/or quantifying theexpression of the reporter gene may also be provided.

For further information and guidance on the design, construction and useof such systems or components thereof which may be adapted for use inpracticing the subject invention, reference to the followingpublications is suggested: Spencer et al, 1993, supra; Rivera et al,1996, supra; Spencer et al, 1996, Current Biology 6, 839–847; Luo et al,1996, Nature, 383, 181–185; Ho et al, 1996, Nature 382, 822–826; Belshawet al, 1996, Proc. Natl. Acad. Sci. USA 93, 4604–4607; Spencer, 1996,TIG 12 (5), 181–187; Spencer et al, 1995, Proc., Natl. Acad. Sci. USA92, 9805–9809; Holsinger et al, 1995, Proc. Natl. Acad. Sci. USA 92,9810–9814; Pruschy et al, 1994, Chemistry & Biology 1 (3), 163–172; andpublished international patent applications WO 94/18317, WO 95/02684, WO95/33052, WO 96/20951, WO 96/41865 and WO 98/02441, the contents of eachof which is incorporated herein by reference.

A key focus of the subject invention is the use of C3 rapalogs asmediators of protein—protein interactions in applications using FKBP andFRB fusion proteins such as described above and elsewhere herein. The C3rapalogs may be used in the various applications of the underlyingdimerization-based technology, including triggering biological events ingenetically engineered cells grown or maintained in culture or presentin whole organisms, including humans and other mammals. The C3 rapalogsmay thus be useful as research reagents in biological experiments invitro, in experiments conducted on animals containing the geneticallyengineered cells, and as prophylactic or therapeutic agents in animaland human health care in subjects containing genetically engineeredcells.

Rapalogs, C3 Rapalogs and Pharmaceutically Acceptable DerivativesThereof

“Rapalogs” as that term is used herein denotes a class of compoundscomprising the various analogs, homologs and derivatives of rapamycinand other compounds related structurally to rapamycin.

Rapalogs include, among others, variants of rapamycin having one or moreof the following modifications relative to rapamycin: demethylation,elimination or replacement of the methoxy at C7, C42 and/or C29;elimination, derivatization or replacement of the hydroxy at C13, C43and/or C28; reduction, elimination or derivatization of the ketone atC14, C24 and/or C30; replacement of the 6-membered pipecolate ring witha 5-membered prolyl ring; and elimination, derivatization or replacementof one or more substituents of the cyclohexyl ring or replacement of thecyclohexyl ring with a substituted or unsubstituted cyclopentyl ring.Rapalogs, as that term is used herein, do not include rapamycin itself,and preferably do not contain an oxygen bridge between C1 and C30.Illustrative examples of rapalogs are disclosed in the documents listedin Table I.

TABLE I WO9710502 WO9418207 WO9304680 U.S. Pat. U.S. Pat. No. 5527907No. 5225403 WO9641807 WO9410843 WO9214737 U.S. Pat. U.S. Pat. No.5484799 No. 5221625 WO9635423 WO9409010 WO9205179 U.S. Pat. U.S. Pat.No. 5457194 No. 5210030 WO9603430 WO94/ U.S. Pat. U.S. Pat. U.S. Pat.04540 No. 5604234 No. 5457182 No. 5208241 WO9600282 WO9402485 U.S. Pat.U.S. Pat. U.S. Pat. No. 5597715 No. 5362735 No. 5200411 WO9516691WO9402137 U.S. Pat. U.S. Pat. U.S. Pat. No. 5583139 No. 5324644 No.5198421 WO9515328 WO9402136 U.S. Pat. U.S. Pat. U.S. Pat. No. 5563172No. 5318895 No. 5147877 WO9507468 WO9325533 U.S. Pat. U.S. Pat. U.S.Pat. No. 5561228 No. 5310903 No. 5140018 WO9504738 WO9318043 U.S. Pat.U.S. Pat. U.S. Pat. No. 5561137 No. 5310901 No. 5116756 WO9504060WO9313663 U.S. Pat. U.S. Pat. U.S. Pat. No. 5541193 No. 5258389 No.5109112 WO9425022 WO9311130 U.S. Pat. U.S. Pat. U.S. Pat. No. 5541189No. 5252732 No. 5093338 WO9421644 WO9310122 U.S. Pat. U.S. Pat. U.S.Pat. No. 5534632 No. 5247076 No. 5091389

Other illustrative rapalogs include those depicted in Table II:

TABLE II

“C3 Rapalogs” are defined above with reference to Formula I and areexemplified by the various classes and subsets of compounds disclosedherein.

The phrase “pharmaceutically acceptable derivative” denotes anypharmaceutically acceptable salt, ester, or salt of such ester, of a C3rapalog, or any other adduct or derivative which, upon administration toa patient, is capable of providing (directly or indirectly) a C3 rapalogas described herein, or a metabolite or residue thereof.Pharmaceutically acceptable derivatives thus include among otherspro-drugs of the rapalogs. A pro-drug is a derivative of a compound,usually with significantly reduced pharmacological activity, whichcontains an additional moiety which is susceptible to removal in vivoyielding the parent molecule as the pharmacologically active species. Anexample of a pro-drug is an ester which is cleaved in vivo to yield acompound of interest. Various pro-drugs of rapamycin and of othercompounds, and materials and methods for derivatizing the parentcompounds to create the pro-drugs, are known and may be adapted to thepresent invention.

The term “aliphatic” as used herein includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, cyclic, orpolycyclic aliphatic hydrocarbons, which are optionally substituted withone or more functional groups. Unless otherwise specified, alkyl, otheraliphatic, alkoxy and acyl groups preferably contain 1–8, and in manycases 1–6, contiguous aliphatic carbon atoms. Illustrative aliphaticgroups thus include, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, —CH₂-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents.

Examples of substituents include: —OH, —OR^(2′), —SH, —SR^(2′), —CHO,═O, —COOH (or ester, carbamate, urea, oxime or carbonate thereof), —NH₂(or substituted amine, amide, urea, carbamate or guanidino derivativetherof), halo, trihaloalkyl, cyano, —SO₂—CF₃, —OSO₂F, —OS(O)₂R¹¹,—SO₂—NHR¹¹, —NHSO₂—R¹¹, sulfate, sulfonate, aryl and heteroarylmoieties. Aryl and heteroaryl substituents may themselves be substitutedor unsubstituted (e.g. mono-, di- and tri-alkoxyphenyl;methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or-phenyl-C(Me)2-CH₂—O—CO—[C3–C6] alkyl or alkylamino).

The term “aliphatic” is thus intended to include alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

As used herein, the term “alkyl” includes both straight, branched andcyclic alkyl groups. An analogous convention applies to other genericterms such as “alkenyl”, “alkynyl” and the like. Furthermore, as usedherein, the language “alkyl”, “alkenyl”, “alkynyl” and the likeencompasses both substituted and unsubstituted groups.

The term “alkyl” refers to groups usually having one to eight,preferably one to six carbon atoms. For example, “alkyl” may refer tomethyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl,sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl tert-pentyl,cyclopentyl, hexyl, isohexyl, cyclohexyl, and the like. Suitablesubstituted alkyls include, but are not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl,hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substitutedbenzyl and the like.

The term “alkenyl” refers to groups usually having two to eight,preferably two to six carbon atoms. For example, “alkenyl” may refer toprop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl,hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. The language“alkynyl,” which also refers to groups having two to eight, preferablytwo to six carbons, includes, but is not limited to, prop-2-ynyl,but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl,hex-5-ynyl, and the like.

The term “cycloalkyl” as used herein refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic or heteroaliphatic or heterocyclic moieties, mayoptionally be substituted.

The term “heteroaliphatic” as used herein refers to aliphatic moietieswhich contain one or more oxygen, sulfur, nitrogen, phosphorous orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched or cyclic and include heterocycles such asmorpholino, pyrrolidinyl, etc.

The term “heterocycle” as used herein refers to cyclic heteroaliphaticgroups and preferably three to ten ring atoms total, includes, but isnot limited to, oxetane, tetrahydrofuranyl, tetrahydropyranyl,aziridine, azetidine, pyrrolidine, piperidine, morpholine, piperazineand the like.

The terms “aryl” and “heteroaryl” as used herein refer to stable mono-or polycyclic, heterocyclic, polycyclic, and polyheterocyclicunsaturated moieties having 3–14 carbon atoms which may be substitutedor unsubstituted. Substituents include any of the previously mentionedsubstituents. Non-limiting examples of useful aryl ring groups includephenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl,alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and thelike. Examples of typical heteroaryl rings include 5-membered monocyclicring groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl,isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like; 6-memberedmonocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazinyl and the like; and polycyclic heterocyclic ring groups such asbenzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, isobenzofuranyl,chromenyl, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl,indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole,tetrahydroquinoline cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl,phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,isothiazolyl, phenothiazinyl, phenoxazinyl, and the like (see e.g.Katritzky, Handbook of Heterocyclic Chemistry). The aryl or heteroarylmoieties may be substituted with one to five members selected from thegroup consisting of hydroxy, C1–C8 alkoxy, C1–C8 branched orstraight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro,halo, trihalomethyl, cyano, and carboxyl. Aryl moieties thus include,e.g. phenyl; substituted phenyl bearing one or more substituentsselected from groups including: halo such as chloro or fluoro, hydroxy,C1–C6 alkyl, acyl, acyloxy, C1–C6 alkoxy (such as methoxy or ethoxy,including among others dialkoxyphenyl moieties such as 2,3-, 2,4-, 2,5-,3,4- or 3,5-dimethoxy or diethoxy phenyl or such asmethylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl; or trisubstitutedphenyl, such as trialkoxy (e.g., 3,4,5-trimethoxy or ethoxyphenyl),3,5-dimethoxy-4-chloro-phenyl, etc.), amino, —SO₂NH₂, —SO₂NH(aliphatic),—SO₂N(aliphatic)₂, —O-aliphatic-COOH, and —O-aliphatic-NH₂ (which maycontain one or two N-aliphatic or N-acyl substituents).

A “halo” substituent according to the present invention may be a fluoro,chloro, bromo or iodo substituent. Fluoro is often the preferredhalogen.

C3 rapalogs may further differ from rapamycin, in addition to themodification at C3, with respect to no, one, two, three, four, five, sixor seven substituent moieties. This class includes among others rapalogswith modifications, relative to rapamycin, at C3 and C13; C3 and C14; C3and a; C3 and C43; C3 and C24; C3 and C28; C3 and C30; C3, C13 and C14;C3, C13 and a; C3, C13 and C43; C3, C13 and C24; C3, C13 and C28; C3,C13 and C30; C3, C14 and a; C3, C14 and C43; C3, C14 and C24; C3, C14and C28; C3, C14 and C30; C3, a and C24; C3, a and C28; C3, a and C30;C3, C24 and C30; C3, C24, C30 and a; C3, C24, C30 and C13; C3, C24, C30and C14; and C3, C24, C30, C13 and a.

One subset of C3 rapalogs of interest in the practice of the variousmethods of the invention are C3 rapalogs in which R^(C30) and R^(C24)are both other than (═O). In certain embodiments of this subset, R^(C30)and R^(C24) are both —OH, e.g. in the “S” configuration. In otherembodiments R^(C30) and R^(C24) are independently selected from OR³.This subset includes among others rapalogs which further differ fromrapamycin with respect to the moiety a. For instance, this subsetincludes compounds of Formula III:

where R^(C3) is other than —H. Alternative substituents for R^(C3) areas disclosed elsewhere herein. Other compounds within this subsetinclude those in which one, two, three, four or five of the hydroxylgroups is epimerized, fluorinated, alkylated, acylated or otherwisemodified via other ester, carbamate, carbonate or urea formation.Illustrative additional compounds for example are those compoundsotherwise as shown in formula III except that the hydroxyl group at C43is in the opposite stereochemical orientation and/or the hydroxyl groupsat C28 and C30 are alkylated, acylated or linked via carbonateformation.

Another subset of C3 rapalogs of special interest are those in which oneor both of R^(C13) and R^(C28) is F. In various embodiments of thissubset, one, two, three, four or five other substituents in formula IIdiffer from the substituents found in rapamycin. For instance, thissubset includes C13 fluoro-C3-rapalogs, C28 fluoro-C3-rapalogs and C13,C28-difluoro-C3-rapalogs.

Another subset of C3 rapalogs of special interest are those in whichR^(C14) is other than O, OH or H, e.g., C3 rapalogs in which R^(C14) is—OR⁶, —NR⁶, NC(O)R⁶, —OC(O)R⁶ or —OC(O)NR⁶, with or without one or moreother modifications relative to rapamycin.

Another subset of C3 rapalogs of interest are those in which R^(C24) isother than ═O, again, with or without one or more other modifications atother positions relative to rapamycin.

Another subset of C3 rapalogs which is of special interest in practicingthe methods of this invention include those which share thestereoisomerism of rapamycin and in which R^(C7a) is —OMe whereinR^(C30) is not ═O, R^(C24) is not ═O, R^(C13) is not —OH, R^(C14) is not═O and/or R³ and/or R⁴ are not H.

Other C3 rapalogs of interest include those in which R^(C14) is OH.

Furthermore, this invention encompasses C3 rapalogs in which one or moreof the carbon—carbon double bonds at the 1,2, 3, 4 or 5,6 positions inrapamycin are saturated, alone or in combination with a modificationelsewhere in the molecule, e.g. at one or more of C13, C43, C24 C28and/or C30. It should also be appreciated that the C3,C4 double bond maybe epoxidized; that the C6 methyl group may be replaced with —CH₂OH or—CH₂OMe; that the C43 hydroxy may be converted to F, Cl or H or othersubstituent; and that the C42 methoxy moiety may be demethylated, in anyof the compounds disclosed herein, using methods known in the art.Likewise, moiety “a” may be replaced with any of the following

Synthetic Guidance

The production of rapamycin by fermentation and by total synthesis isknown. The production of a number of rapalogs (other than C3 rapalogs)by fermentation or synthetic modification of fermentation products isalso known. These include among others rapalogs bearing alternativemoieties to the characteristic cyclohexyl ring or pipecolate ring ofrapamycin, as well as C29-desmethyl-rapamycin andC29-desmethoxyrapamycin and a variety of other rapalogs such as aredisclosed herein.

Rapamycin may be converted into a C3 rapalog using silyl ethers asdisclosed in the experimental examples which follow.

The C3 rapalogs can be produced using a variety of techniques. Thesetechniques can be synthetic processes involving nucleophilic reagentsand catalysts. A first rapalog can be treated with a nucleophilicreagent under conditions which allow for the formation of the desired C3rapalog. The first rapalog can be a C7 modified rapalog, e.g. C7-methoxyrapalog. These conditions include such factors as type of nucleophilicreagent, type of catalysts and selection of purification technique usedto isolate or purify the final C3 rapalog.

For example, substituted rapalogs of the invention can be prepared bytreating a rapalog having a substituent attached to the macrocyclicring, e.g., at the C7 position, with a nucleophilic reagent, e.g., asilyl ether, in the presence of a catalyst. In another embodiment,rapamycin can be converted into C3 rapalogs using silyl enol ethersknown to those skilled in the art.

Suitable substituent include those recognized in the art by skilledartisans and include halogens, tosylates, mesylates, esters andalkoxides, and preferably lower alkyl alkoxides. It was discovered thattreatment of rapalogs having a substituent at the C7 position withsuitable nucleophiles formed a rapalog modified at the C3 position bythe nucleophile.

Suitable nucleophilic reagents include those recognized in the art byskilled artisans and include silyl ethers, silyl enol ethers, grignardreagents, enolates, carbanions and the like. A preferred nucleophilicreagent is a silyl ether, preferably trialkylsilylethers, particularlysubstituted and unsubstituted allyl groups, e.g.,methallyltrialkylsilylether.

The term “silyl ether” is art recognized and is intended to includethose compounds having the formula RC3-SiRR′R″ (2) wherein R, R′ and R″are aliphatic, aromatic, heteroaliphatic or heteroaromatic moieties andRC3 is as defined above, preferably a substituted or unsubstituted allylmoiety. Moreover, silyl ethers are commercially available and can bepurchased from chemical supply companies such as ALDRICH®, and SIGMA®.

The term “silyl enol ether” is art recognized and is intended to includethose compounds having the formula

wherein R, R′ and R″ are aliphatic, aromatic, heteroaliphatic orheteroaromatic moieties and RC3 is as defined above. Silyl enol ethersare also commercially available and can be purchased from chemicalsupply companies such as ALDRICH®, and SIGMA®.

Suitable catalysts include Lewis acid catalysts such as AlCl₃, ZnCl₂,p-toluene sulfonic acid, and preferably BF₃ etherate.

The following reaction scheme depicted below provides an example ofnucleophilic displacement of a leaving group by a nucleophilic reagent,e.g., a silyl ether. Treatment of compound 1, a rapalog,

with silyl ether, 2, under conditions which permit the formation of acompound having formula 3 include combining a C7 heteroatom bearingrapalog, e.g., OMe, with a trialkylsilylether in the presence of acatalyst, preferably BF₃/etherate. In general, the reaction conditionsare performed at ambient temperature or below, preferably at about −40°C.

C3 rapalogs comprising additional modifications with respect to thestructure of rapamycin may be prepared analogously, starting with thecorresponding rapalog in place of rapamycin. Alternatively, one or moreadditional transformations may be carried out on a C3 rapalogintermediate.

Methods and materials for effecting various chemical transformations ofrapamycin and structurally related macrolides are known in the art, asare methods for obtaining rapamycin and various rapalogs byfermentation. Many such chemical transformations of rapamycin andvarious rapalogs are disclosed in the patent documents identified inTable I, above, which serve to illustrate the level of skill andknowledge in the art of chemical synthesis and product recovery,purification and formulation which may be applied in practicing thesubject invention. The following representative transformations and/orreferences which can be employed to produce the desired rapalogs areillustrative:

ring position modified literature reference C-13 C13−>F: protect C28 andC43, rxn at 0° C. C-14 Schubert, et al. Angew Chem Int Ed Engl 23, 167(1984). C-20 Nelson, U.S. Pat. No. 5,387,680 C-24 U.S. Pat. No.5,373,014; 5,378,836 Lane, et al. Synthesis 1975, p136. C-30 Luengo etal. Tet. Lett. 35, 6469 (1994) various Or et al, U.S. Pat. Nos.5,527,907 and 5,583,139 positions Luengo, WO 94/02136; Cottens et al, WO95/16691 WO 98/02441 (Holt et al)

Additionally, it is contemplated that rapalogs for use in this inventionas well as intermediates for the production of such rapalogs may beprepared by directed biosynthesis, e.g. as described by Katz et al, WO93/13663 and by Cane et al, WO 9702358.

Novel rapalogs of this invention may be prepared by one of ordinaryskill in this art relying upon methods and materials known in the art asguided by the disclosure presented herein. For instance, methods andmaterials may be adapted from known methods set forth or referenced inthe documents cited above, the full contents of which are incorporatedherein by reference. Additional guidance and examples are providedherein by way of illustration and further guidance to the practitioner.It should be understood that the chemist of ordinary skill in this artwould be readily able to make modifications to the foregoing, e.g. toadd appropriate protecting groups to sensitive moieties duringsynthesis, followed by removal of the protecting groups when no longerneeded or desired, and would be readily capable of determining othersynthetic approaches.

Purification of mixtures of rapalogs isolated from the above describedconditions can be accomplished using a technique which allows for theisolation or recovery of the desired C3 rapalog, e.g., a desired purityor in a desired form, e.g., substantially free of C7 rapalog. An exampleof such a technique is the recycling HPLC technique described in theexamples below. Use of recycling HPLC lead to the discovery, under theconditions described above, that nucleophiles add to the C3 positionwith concomitant displacement of a C7 group to yield C3 rapalogs.Structural confirmation that the rapalogs were functionalized at the C3position was determined by mass spectral analysis and/or NMRspectroscopy.

As a non-limiting example, an unpurified reaction mixture of rapalogproducts can be eluted through an HPLC column and recycled back throughthe column to increase separation of components and thus, purity of thefinal isolates. HPLC systems are known to those skilled in the art canbe adapted to include such recycling features. Generally, the eluant andthe component(s) are recycled until a desired separation of componentsand purity is achieved, e.g., 15 to 20 cycles. One skilled in the artcan determine how many cycles are required, the solvent(s) required, thetype of column, etc. to achieve the purity desired.

For example, a polystyrene size exclusion column can be used. Thepolystyrene column can be functionalized, e.g., contains hydroxylfunctionality, to facilitate separation of component. In oneparticularly preferred embodiment, a JAIGEL GS-310 column can be used toeffect separation of components.

One aspect of the invention pertains to the purity of the isolatedrapalogs. Purity of the isolate rapalogs can be determined by techniquesknown to those skilled in the art and include mass spectrometry, ¹H NMRand ¹³C NMR. In one embodiment, the isolated C3 rapalogs or theinvention are substantially free contaminants, e.g., substantially freeof C7 rapalogs. One example of determining the purity of the isolated C3rapalog is by ¹H NMR. The purity of the compound can be determined byanalyzing the peak height ratio of the desired product to that ofcontaminants. In one embodiment, the ratio is at least about 5:1(product/contaminant). In another embodiment, the ratio is at leastabout 10:1, more preferably 50:1, most preferably 100:1 with the mostpreferred being that no contaminants are detected by NMR.

In another aspect, purity of the isolated rapalog can be determined by¹H NMR by analyzing the ratio between the peak heights associated withthose groups attached at the C7 position of the starting material tothat of the peak heights associated with the groups attached at the C3position in the resultant product. The purity of the C3 rapalog can bedetermined by analyzing the peak height ratio of the desired product tothat of peaks associated with the starting material containing a C7group. In one embodiment, the ratio is at least about 5:1 (isolated C3rapalog product/C7 starting material). In another embodiment, the ratiois at least about 10:1, more preferably 50:1, most preferably 100:1 withthe most preferred being that no contaminants are detected by NMR.

In another embodiment, the isolated C3 rapalog is at least about 90%pure, as determined by analytical techniques known in the art, e.g.capillary gas chromatography, analytical HPLC, etc. In a more preferredembodiment, the isolated C3 rapalog is at least about 95% pure. Inanother embodiment, the isolated C3 rapalog is at least about 98% pure.In a most preferred embodiment, the isolated C3 rapalog is about 100%pure.

FKBP Domains and Fusion Proteins

The FKBP fusion protein comprises at least one FKBP domain containingall or part of the peptide sequence of an FKBP domain and at least oneheterologous action domain. This chimeric protein must be capable ofbinding to a C3 rapalog of this invention, preferably with a Kd valuebelow about 100 nM, more preferably below about 10 nM and even morepreferably below about 1 nM, as measured by direct binding measurement(e.g. fluorescence quenching), competition binding measurement (e.g.versus FK506), inhibition of FKBP enzyme activity (rotamase), or otherassay methodology. Typically the chimeric protein will contain one ormore protein domains comprising peptide sequence selected from that of anaturally occurring FKBP protein such as human FKBP12, e.g. as describedin International Patent Application PCT/US94/01617. That peptidesequence may be modified to adjust the binding specificity, usually withreplacement, insertion or deletion of 10 or fewer, preferably 5 orfewer, amino acid residues. Such modifications are elected in certainembodiments to yield one or both of the following binding profiles: (a)binding of a C3 rapalog to the modified FKBP domain, or chimeracontaining it, preferably at least one, and more preferably at leasttwo, and even more preferably three or four or more, orders of magnitudebetter (by any measure) than to FKBP12 or the FKBP endogenous to thehost cells to be engineered; and (b) binding of the FKBP:rapalog complexto the FRB fusion protein, preferably at least one, and more preferablyat least two, and even more preferably at least three, orders ofmagnitude better (by any measure) than to the FRAP or otherFRB-containing protein endogenous to the host cell to be engineered.

The FKBP chimera also contains at least one heterologous action domain,i.e., a protein domain containing non-FKBP peptide sequence. The actiondomain may be a DNA-binding domain, transcription activation domain,cellular localization domain, intracellular signal transduction domain,etc., e.g. as described elsewhere herein or in PCT/US94/01617 or theother cited references. Generally speaking, the action domain is capableof directing the chimeric protein to a selected cellular location or ofinitiating a biological effect upon association or aggregation withanother action domain, for instance, upon multimerization of proteinscontaining the same or different action domains.

A recombinant nucleic acid encoding such a fusion protein will becapable of selectively hybridizing to a DNA encoding the parent FKBPprotein, e.g. human FKBP 12, or would be capable of such hybridizationbut for the degeneracy of the genetic code. Since these chimericproteins contain an action domain derived from another protein, e.g.Gal4, VP16, FAS, CD3 zeta chain, etc., the recombinant DNA encoding thechimeric protein will also be capable of selectively hybridizing to aDNA encoding that other protein, or would be capable of suchhybridization but for the degeneracy of the genetic code.

FKBP fusion proteins of this invention, as well as FRB fusion proteinsdiscussed in further detail below, may contain one or more copies of oneor more different ligand binding domains and one or more copies of oneor more action domains. The ligand binding domain(s) (i.e., FKBP and FRBdomains) may be N-terminal, C-terminal, or interspersed with respect tothe action domain(s). Embodiments involving multiple copies of a ligandbinding domain usually have 2, 3 or 4 such copies. For example, an FKBPfusion protein may contain 2, 3 or 4 FKBP domains. The various domainsof the FKBP fusion proteins (and of the FRB fusion proteins discussedbelow) are optionally separated by linking peptide regions which may bederived from one of the adjacent domains or may be heterologous.

Illustrative examples of FKBP fusion proteins useful in the practice ofthis invention include the FKBP fusion proteins disclosed inPCT/US94/01617 (Stanford & Harvard), PCT/US94/08008 (Stanford &Harvard), Spencer et al (supra), PCT/US95/10591 (ARIAD), PCT/US95/06722(Mitotix, Inc.) and other references cited herein; the FKBP fusionproteins disclosed in the examples which follow; variants of any of theforegoing FKBP fusion proteins which contain up to 10 (preferably 1–5)amino acid insertions, deletions or substitutions in one or more of theFKBP domains and which are still capable of binding to rapamycin or to arapalog; variants of any of the foregoing FKBP fusion proteins whichcontain one or more copies of an FKBP domain which is encoded by a DNAsequence capable of selectively hybridizing to a DNA sequence encoding anaturally occurring FKBP domain and which are still capable of bindingto rapamycin or to a rapalog; variants of any of the foregoing in whichone or more heterologous action domains are deleted, replaced orsupplemented with a different heterologous action domain; variants ofany of the foregoing FKBP fusion proteins which are capable of bindingto rapamycin or a rapalog and which contain an FKBP domain derived froma non-human source; and variants of any of the foregoing FKBP fusionproteins which contain one or more amino acid residues corresponding toTyr26, Phe36, Asp37, Arg42, Phe46, Phe48, Glu54, Val55, or Phe99 ofhuman FKBP12 in which one or more of those amino acid residues isreplaced by a different amino acid, the variant being capable of bindingto rapamycin or a rapalog.

For instance, in a number of cases the FKBP fusion proteins comprisemultiple copies of an FKBP domain containing amino acids 1–107 of humanFKBP12, separated by the 2-amino acid linker Thr-Arg encoded by ACTAGA,the ligation product of DNAs digested respectively with the restrictionendonucleases SpeI and XbaI. The following table provides illustrativesubsets of mutant FKBP domains based on the foregoing FKBP12 sequence:

Illustrative Mutant FKBPs F36A Y26V F46A W59A F36V Y26S F48H H87W F36MD37A F48L H87R F36S I90A F48A F36V/F99A F99A I91A E54A F36V/F99G F99GF46H E54K F36M/F99A Y26A F46L V55A F36M/F99G note: Entries identify thenative amino acid by single letter code and sequence position, followedby the replacement amino acid in the mutant. Thus, F36V designates ahuman FKBP12 sequence in which phenylalanine at position 36 is replacedby valine. F36V/F99A indicates a double mutation in which phenylalanineat positions 36 and 99 are replaced by valine and alanine, respectively.@FRB Domains and Fusion Proteins

The FRB fusion protein comprises at least one FRB domain (which maycomprise all or part of the peptide sequence of a FRAP protein or avariant thereof, as described elsewhere) and at least one heterologouseffector domain.

Generally speaking, the FRB domain, or a chimeric protein encompassingit, is encoded by a DNA molecule capable of hybridizing selectively to aDNA molecule encoding a protein comprising a naturally occurring FRBdomain, e.g. a DNA molecule encoding a human or other mammalian FRAPprotein or one of yeast proteins, Tor-1 or Tor-2 or the previouslymentioned Candida FRB-containing protein. FRB domains of this inventioninclude those which are capable of binding to a complex of an FKBPprotein and a C3 rapalog of this invention.

The FRB fusion protein must be capable of binding to the complex formedby the FKBP fusion protein with a C3 rapalog of this invention.Preferably, the FRB fusion protein binds to that complex with a Kd valuebelow 200 μM, more preferably below 10 μM, as measured by conventionalmethods. The FRB domain will be of sufficient length and composition tomaintain high affinity for a complex of the rapalog with the FKBP fusionprotein. In some embodiments the FRB domain spans fewer than about 150amino acids in length, and in some cases fewer than about 100 aminoacids. One such region comprises a 133 amino acid region of human FRAPextending from Val2012 through Tyr2144. See Chiu et al, 1994, Proc.Natl. Acad. Sci. USA 91:12574–12578. An FRB region of particularinterest spans Glu2025 through Gln2114 of human FRAP and retainsaffinity for a FKBP12-rapamycin complex or for FKBP-rapalog complex. Insome embodiments Q2214 is removed from the 90-amino acid sequencerendering this an 89-amino acid FRB domain. The FRB peptide sequence maybe modified to adjust the binding specificity, usually with replacement,insertion or deletion, of 10 or fewer, preferably 5 or fewer, aminoacids. Such modifications are elected in certain embodiments to achievea preference towards formation of the complex comprising one or moremolecules of the FKBP fusion protein, FRB fusion protein and a C3rapalog over formation of complexes of endogenous FKBP and FRAP proteinswith the rapalog. Preferably that preference is at least one, and morepreferably at least two, and even more preferably three, orders ofmagnitude (by any measure).

A recombinant DNA encoding such a protein will be capable of selectivelyhybridizing to a DNA encoding a FRAP species, or would be capable ofsuch hybridization but for the degeneracy of the genetic code. Again,since these chimeric proteins contain an effector domain derived fromanother protein, e.g. Gal4, VP 16, Fas, CD3 zeta chain, etc., therecombinant DNA encoding the chimeric protein will be capable ofselectively hybridizing to a DNA encoding that other protein, or wouldbe capable of such hybridization but for the degeneracy of the geneticcode.

Illustrative examples of FRB chimeras useful in the practice of thisinvention include those disclosed in the examples which follow, variantsthereof in which one or more of the heterologous domains are replacedwith alternative heterologous domains or supplemented with one or moreadditional heterologous domains, variants in which one or more of theFRB domains is a domain of non-human peptide sequence origin (such asTor 2 or Candida for example), and variants in which the FRB domain ismodified by amino acid substitution, replacement or insertion asdescribed herein, so long as the chimera is capable of binding to acomplex formed by an FKBP protein and a C3 rapalog of this invention. Anillustrative FRB fusion protein contains one or more FRBs of at least89-amino acids, containing a sequence spanning at least residues2025–2113 of human FRAP, separated by the linker Thr-Arg formed byligation of SpeI-XbaI sites as mentioned previously. It should beappreciated that such restriction sites or linkers in any of the fusionproteins of this invention may be deleted, replaced or extended usingconventional techniques such as site-directed mutagenesis.

Mixed Chimeric Proteins

A third type of chimeric protein comprises one or more FKBP domains, oneor more heterologous effector domains, and one or more FRB domains asdescribed for the FRB fusion proteins.

Mixed chimeric protein molecules are capable of forming homodimeric orhomomultimeric protein complexes in the presence of a C3 rapalog towhich they bind. Embodiments involving mixed chimeras have the advantageof requiring the introduction into cells of a single recombinant nucleicacid construct in place of two recombinant nucleic acid constructsotherwise required to direct the expression of both an FKBP fusionprotein and a FRB fusion protein.

A recombinant DNA encoding a mixed chimeric protein will be capable ofselectively hybridizing to a DNA encoding an FKBP protein, a DNAencoding FRAP, and a heterologous DNA sequence encoding the protein fromwhich one or more effector domains is derived (e.g. Gal4, VP16, Fas, CD3zeta chain, etc.), or would be capable of such hybridization but for thedegeneracy of the genetic code.

Heterologous Domains

As mentioned above, the heterologous effector domains of the FKBP andFRB fusion proteins are protein domains which, upon mutual associationof the chimeric proteins bearing them, are capable of triggering (orinhibiting) DNA-binding and/or transcription of a target gene; actuatingcell growth, differentiation, proliferation or apoptosis; directingproteins to a particular cellular location; or actuating otherbiological events.

Embodiments involving regulatable gene transcription involve the use oftarget gene constructs which comprise a target gene (which encodes apolypeptide, antisense RNA, ribozyme, etc. of interest) under thetranscriptional control of a DNA element responsive to the associationor multimerization of the heterologous domains of the 1st and 2dchimeric proteins.

In embodiments of the invention involving direct activation oftranscription, the heterologous domains of the 1 st and 2d chimericproteins comprise a DNA binding domain such as Gal4 or a chimeric DNAbinding domain such as ZFHD1, discussed below, and a transcriptionalactivating domain such as those derived from VP16 or p65, respectively.The multimerization of a chimeric protein containing such atranscriptional activating domain to a chimeric protein containing a DNAbinding domain targets the transcriptional activator to the promoterelement to which the DNA binding domain binds, and thus activates thetranscription of a target gene linked to that promoter element.Foregoing the transcription activation domain or substituting arepressor domain (see PCT/US94/01617) in place of a transcriptionactivation domain provides an analogous chimera useful for inhibitingtranscription of a target gene. Composite DNA binding domains and DNAsequences to which they bind are disclosed in Pomerantz et al, 1995,supra, the contents of which are incorporated herein by reference. Suchcomposite DNA binding domains may be used as DNA binding domains in thepractice of this invention, together with a target gene constructcontaining the cognate DNA sequences to which the composite DBD binds.

In embodiments involving indirect activation of transcription, theheterologous domains of the chimeras are effector domains of signalingproteins which upon aggregation or multimerization trigger theactivation of transcription under the control of a responsive promoter.For example, the signaling domain may be the intracellular domain of thezeta subunit of the T cell receptor, which upon aggregation, triggerstranscription of a gene linked to the IL-2 promoter or a derivativethereof (e.g. iterated NF-AT binding sites).

In another aspect of the invention, the heterologous domains are proteindomains which upon mutual association are capable of triggering celldeath. Examples of such domains are the intracellular domains of the Fasantigen or of the TNF R1. Chimeric proteins containing a Fas domain canbe designed and prepared by analogy to the disclosure of PCT/US94/01617.

Engineered Receptor Domains

As noted previously, the FKBP and FRB domains may contain peptidesequence selected from the peptide sequences of naturally occurring FKBPand FRB domains. Naturally occurring sequences include those of humanFKBP12 and the FRB domain of human FRAP. Alternatively, the peptidesequences may be derived from such naturally occurring peptide sequencesbut contain generally up to 10, and preferably 1–5, mutations in one orboth such peptide sequences. As disclosed in greater detail elsewhereherein, such mutations can confer a number of important features. Forinstance, an FKBP domain may be modified such that it is capable ofbinding a C3 rapalog preferentially, i.e. at least one, preferably two,and even more preferably three or four or more orders of magnitude moreeffectively, with respect to rapalog binding by the unmodified FKBPdomain. An FRB domain may be modified such that it is capable of bindinga (modified or unmodified) FKBP:rapalog complex preferentially, i.e. atleast one, preferably two, and even more preferably three orders ofmagnitude more effectively, with respect to the unmodified FRB domain.FKBP and FRB domains may be modified such that they are capable offorming a tripartite complex with a C3 rapalog, preferentially, i.e. atleast one, preferably two, and even more preferably three orders ofmagnitude more effectively, with respect to unmodified FKBP and FRBdomains.

(a) FKBP

Methods for identifying FKBP mutations that confer enhanced ability tobind derivatives of FK506 containing various substituents (“bumps”) weredisclosed in PCT/US94/01617. Similar strategies can be used to obtainmodified FKBPs that preferentially bind bumped rapamycin derivatives,i.e., rapalogs. The structure of the complex between rapamycin andFKBP12 is known (see for example Van Duyne et al., J. Am. Chem. Soc.(1991) 113, 7433–7434). Such data can be used to reveal amino acidresidues that would clash with various rapalog substituents. In thisapproach, molecular modeling is used to identify candidate amino acidsubstitutions in the FKBP domain that would accommodate the rapalogsubstituent(s), and site-directed mutagenesis may then be used toengineer the protein mutations so identified. The mutants are expressedby standard methods and their binding affinity for the rapalogsmeasured, for example by inhibition of rotamase activity, or bycompetition for binding with a molecule such as FK506, if the mutantretains appropriate activity/affinity.

More particularly, not to be bound by theory, we contemplate thatcertain C3 rapalogs of this invention, e.g. rapalogs with modificationsrelative to rapamycin at C-13 or C-14 bind preferentially to FKBPs inwhich one or more of the residues, Tyr26, Phe36, Asp37, Tyr82 and Phe99,are substituted with amino acids that have smaller side chains (such asGly, Ala, Val, Met and Ser). Examples of mutant FKBPs with modificationsat positions 26 or 36 are noted in the “Illustrative Mutant FKBPs” tableabove. Similarly, we contemplate that rapalogs with modifications at C20(i.e., rapalogs in which R4 is other than —H) bind preferentially toFKBPs in which Tyr82 and/or Ile56 are replaced by other amino acids,especially those with smaller side chains. In a further example, wecontemplate that rapalogs bearing modifications at C24 (i.e., in which Wis other than ═O) bind preferentially to FKBPs in which one or more ofPhe46, Phe48 and Val55 are replaced by other amino acids, againespecially those with smaller side chains. Moreover, we envisage thatrapalogs with modifications at C28 and/or C30 (i.e., in which R3 isother than H and/or V is other than ═O) bind preferentially to FKBPs inwhich Glu54 is replaced by another amino acid, especially one with asmaller side chain. In all of the above examples, single or multipleamino acid substitutions may be made. Again, specific examples are notedin the previous table.

An alternative to iterative engineering and testing of single ormultiple mutants is to co-randomize structurally-identified residuesthat are or would be in contact with or near one or more rapalog orrapamycin substituents. A collection of polypeptides containing FKBPdomains randomized at the identified positions (such as are noted in theforegoing paragraph) is prepared e.g. using conventional synthetic orgenetic methods. Such a collection represents a set of FKBP domainscontaining replacement amino acids at one or more of such positions. Thecollection is screened and FKBP variants are selected which possess thedesired rapalog binding properties. In general, randomizing severalresidues simultaneously is expected to yield compensating mutants ofhigher affinity and specificity for a given bumped rapalog as itmaximizes the likelihood of beneficial cooperative interactions betweensidechains. Techniques for preparing libraries randomized at discretepositions are known and include primer-directed mutagenesis usingdegenerate oligonucleotides, PCR with degenerate oligonucleotides, andcassette mutagenesis with degenerate oligonucleotides (see for exampleLowman, H. B, and Wells, J. A. Methods: Comp. Methods Enzymol. 1991. 3,205–216; Dennis, M. S. and Lazarus, R. A. 1994. J. Biol. Chem. 269,22129–22136; and references therein).

We further contemplate that in many cases, randomization of only the fewresidues in or near direct contact with a given position in rapamycinmay not completely explore all the possible variations in FKBPconformation that could optimally accommodate a rapalog substituent(bump). Thus the construction is also envisaged of unbiased librariescontaining random substitutions that are not based on structuralconsiderations, to identify subtle mutations or combinations thereofthat confer preferential binding to bumped rapalogs. Several suitablemutagenesis schemes have been described, including alanine-scanningmutagenesis (Cunningham and Wells (1989) Science 244, 1081–1085), PCRmisincorporation mutagenesis (see e.g. Cadwell and Joyce, 1992 PCR Meth.Applic. 2, 28–33), and ‘DNA shuffling’ (Stemmer, 1994, Nature 370,389–391 and Crameri et al, 1996, Nature Medicine 2, 100–103). Thesetechniques produce libraries of random mutants, or sets of singlemutants, that are then searched by screening or selection approaches.

In many cases, an effective strategy to identify the best mutants forpreferential binding of a given bump is a combination of structure-basedand unbiased approaches. See Clackson and Wells, 1994, TrendsBiotechnology 12, 173–184 (review). For example we contemplate theconstruction of libraries in which key contact residues are randomizedby PCR with degenerate oligonucleotides, but with amplificationperformed using error-promoting conditions to introduce furthermutations at random sites. A further example is the combination ofcomponent DNA fragments from structure-based and unbiased randomlibraries using DNA shuffling.

Screening of libraries for desirable mutations may be performed by useof a yeast 2-hybrid system (Fields and Song (1989) Nature 340, 245–246).For example, an FRB-VP16 fusion may be introduced into one vector, and alibrary of randomized FKBP sequences cloned into a separate GAL4 fusionvector. Yeast co-transformants are treated with rapalog, and thoseharboring complementary FKBP mutants are identified by for examplebeta-galactosidase or luciferase production (a screen), or survival onplates lacking an essential nutrient (a selection), as appropriate forthe vectors used. The requirement for bumped rapamycin to bridge theFKBP-FRAP interaction is a useful screen to eliminate false positives.

A further strategy for isolating modified ligand-binding domains fromlibraries of FKBP (or FRB) mutants utilizes a genetic selection forfunctional dimer formation described by Hu et. al. (Hu, J. C., et al.1990. Science. 250:1400–1403; for review see Hu, J. C. 1995. Structure.3:431–433). This strategy utilizes the fact that the bacteriophagelambda repressor cI binds to DNA as a homodimer and that binding of suchhomodimers to operator DNA prevents transcription of phage genesinvolved in the lytic pathway of the phage life cycle. Thus, bacterialcells expressing functional lambda repressor are immune to lysis bysuperinfecting phage lambda. Repressor protein comprises an aminoterminal DNA binding domain (amino acids 1–92), joined by a 40 aminoacid flexible linker to a carboxy terminal dimerization domain. Theisolated N-terminal domain binds to DNA with low affinity due toinefficient dimer formation. High affinity DNA binding can be restoredwith heterologous dimerization domains such as the GCN4 “leucinezipper”. Hu et al have described a system in which phage immunity isused as a genetic selection to isolate GCN4 leucine zipper mutantscapable of mediating lambda repressor dimer formation from a largepopulation of sequences (Hu et. al., 1990).

For example, to use the lambda repressor system to identify FRAP mutantscomplementary to bumped rapalogs, lambda repressor-FRAP librariesbearing mutant FRAP sequences are transformed into E. coli cellsexpressing wild-type lambda repressor-FKBP protein. Plasmids expressingFRAP mutants are isolated from those colonies that survive lysis onbacterial plates containing high titres of lambda phage and “bumped”rapamycin compounds. Alternatively, to isolate FKBP mutants, the abovestrategy is repeated with lambda repressor-FKBP libraries bearing mutantFKBP sequences transformed into E. coli cells expressing wildtype lambdarepressor-FRAP protein.

A further alternative is to clone the randomized FKBP sequences into avector for phage display, allowing in vitro selection of the variantsthat bind best to the rapalog. Affinity selection in vitro may beperformed in a number of ways. For example, rapalog is mixed with thelibrary phage pool in solution in the presence of recombinant FRAPtagged with an affinity handle (for example a hexa-histidine tag, orGST), and the resultant complexes are captured on the appropriateaffinity matrix to enrich for phage displaying FKBP harboringcomplementary mutations. Techniques for phage display have beendescribed, and other in vitro selection systems can also be contemplated(for example display on lambda phage, display on plasmids, display onbaculovirus). Furthermore, selection and screening strategies can alsobe used to improve other properties of benefit in the application ofthis invention, such as enhanced stability in vivo. For a review seeClackson, T. & Wells, J. A. 1994. Trends Biotechnol. 12, 173–184.

(b) FRAP

Similar considerations apply to the generation of mutant FRB domainswhich bind preferentially to C3 rapalogs containing modifications (i.e.,are ‘bumped’) relative to rapamycin in the FRAP-binding portion of themacrocycle. For example, one may obtain preferential binding usingrapalogs bearing substituents other than —OMe at the C7 position withFRBs based on the human FRAP FRB peptide sequence but bearing amino acidsubstitutions for one of more of the residues Tyr2038, Phe2039, Thr2098,Gln2099, Trp2101, Ser2035, Tyr2105, Pro2095, and any other residue inthe vicinity of the rapamycin, triene or residues near the rapamycintriene and Asp2102. Exemplary mutations include Y2038H, Y2038L, Y2038V,Y2038A, F2039H, F2039L, F2039A, F2039V, D2102A, T2098A, T2098N, andT2098S. Rapalogs bearing substituents other than —OH at C28 and/orsubstituents other than ═O at C30 may be used to obtain preferentialbinding to FRAP proteins bearing an amino acid substitution for Glu2032.Exemplary mutations include E2032A and E2032S. Proteins comprising anFRB containing one or more amino acid replacements at the foregoingpositions, libraries of proteins or peptides randomized at thosepositions (i.e., containing various substituted amino acids at thoseresidues), libraries randomizing the entire protein domain, orcombinations of these sets of mutants are made using the proceduresdescribed above to identify mutant FRAPs that bind preferentially tobumped rapalogs.

The affinity of candidate mutant FRBs for the complex of an FKBP proteincomplexed with a rapalog may be assayed by a number of techniques; forexample binding of in vitro translated FRB mutants to GST-FKBP in thepresence of drug (Chen et al. 1995. Proc. Natl. Acad. Sci. USA 92,4947–4951); or ability to participate in a rapalog-dependenttranscriptionally active complex with an appropriate FKBP fusion proteinin a yeast or mammalian two- three-hybrid assay.

FRB mutants with desired binding properties may be isolated fromlibraries displayed on phage using a variety of sorting strategies. Forexample, a rapalog is mixed with the library phage pool in solution inthe presence of recombinant FKBP tagged with an affinity handle (forexample a hexa-histidine tag, or GST), and the resultant complexes arecaptured on the appropriate affinity matrix to enrich for phagedisplaying FRAP harboring complementary mutations.

An additional feature of the FRB fusion protein that may vary in thevarious embodiments of this invention is the exact sequence of the FRBdomain used. In some applications it may be preferred to use portions ofan FRB which are larger than the minimal (89 amino acid) FRB domain.These include extensions N-terminal to residue Glu2025 (preferablyextending to at least Arg2018 or Ile2021), as well as C-terminalextensions beyond position 2113, e.g. to position 2113, 2141 or 2174 orbeyond), which may in some cases improve the stability of the folded FRBdomain and/or the efficiency of expression. Other applications in whichdifferent FRB sequence termini may be used include those in which a longlinker is desired for steric reasons on one or both sides of the FRBdomain, for example to accommodate the distortions of the polypeptidechain required for FRB-mediated protein—protein association at the cellmembrane or on DNA. Conversely, in other applications short linkers onone or both sides of the FRB domain may be preferred or required topresent the heterologous effector domain(s) appropriately for biologicalfunction. In human gene therapy applications the use of naturallyoccurring human FRAP sequence for such linkers will generally bepreferred to the introduction of heterologous sequences, or reduce therisk of provoking an immune response in the host organism.

Some rapalogs, especially rapalogs with modifications or substituents(relative to rapamycin) at positions believed to lie near the boundarybetween the FKBP binding domain and the FRAP binding domain, such asthose on C28, C30, C7 and C24, possess reduced ability, relative torapamycin, to form complexes with both mammalian FKBP and FRB domains,in particular, with those domains containing naturally occurring humanpeptide sequence. That reduced ability may be manifested as a reducedbinding affinity as determined by any of the direct or indirect assaymeans mentioned herein or as reduced immunosuppressive activity asdetermined in an appropriate assay such as a T cell proliferation assay.In such cases, iterative procedures may be used to identify pairs ofmutant FKBPs and mutant FRBs that are capable of complexing with therapalog more effectively than the corresponding domains containingnaturally occurring human peptide sequence. For example, one may firstidentify a complementary modified FKBP domain capable of binding to therapalog, as discussed previously, and then using this mutant FKBP domainas an affinity matrix in complex with the rapalog, one may select acomplementary modified FRB domain capable of associating with thatcomplex. Several cycles of such mutagenesis and screening may beperformed to optimize the protein pair.

For some embodiments, it will be desirable to use FRB and/or FKBPdomains containing mutations that can affect the protein—proteininteraction. For instance, mutant FKBP domains which when bound to agiven rapalog are capable of complexing with an endogenous FRBmeasurably less effectively than to a mutant FRB are of particularinterest. Also of interest are mutant FRB domains which are capable ofassociating with a complex of a mutant FKBP with a given rapalogmeasurable more effectively than with a complex of an endogenous FKBPwith the rapalog. Similar selection and screening approaches to thosedelineated previously can be used (i) to identify amino acidsubstitutions, deletions or insertions to an FKBP domain whichmeasurably diminish the domain's ability to form the tripartite complexwith a given rapalog and the endogenous FRB; (ii) to identify amino acidsubstitutions, deletions or insertions to an FRB domain which measurablydiminish the domain's ability to form the tripartite complex with agiven rapalog and the endogenous FKBP; and (iii) to select and/orotherwise identify compensating mutation(s) in the partner protein. Asexamples of suitable mutant FKBPs with diminished effectiveness intripartite complex formation, we include mammalian, preferably humanFKBP in which one or both of His87 and Ile90 are replaced with aminoacids such as Arg, Trp, Phe, Tyr or Lys which contain bulky side chaingroups; FRB domains, preferably containing mammalian, and morepreferably of human, peptide sequence may then be mutated as describedabove to generate complementary variants which are capable of forming atripartite complex with the mutant FKBP and a given rapalog.Illustrative FRB mutations which may be useful with H87W or H87RhFKBP12s include human FRBs in which Y2038 is replaced by V, S, A or L;F2039 is replaced by A; and/or R2042 is replaced by L, A or S.Illustrative FRB mutations which may be useful with I90W or I90RhFKBP12s include human FRBs in which K2095 is replaced with L, S, A orT.

Additionally, in optimizing the receptor domains of this invention, itshould be appreciated that immunogenicity of a polypeptide sequence isthought to require the binding of peptides by MHC proteins and therecognition of the presented peptides as foreign by endogenous T-cellreceptors. It may be preferable, at least in human gene therapyapplications, to tailor a given foreign peptide sequence, includingjunction peptide sequences, to minimize the probability of its beingimmunologically presented in humans. For example, peptide binding tohuman MHC class I molecules has strict requirements for certain residuesat key ‘anchor’positions in the bound peptide: e.g. HLA-A2 requiresleucine, methionine or isoleucine at position 2 and leucine or valine atthe C-terminus (for review see Stern and Wiley (1994) Structure 2,145–251). Thus in engineering proteins in the practice of thisinvention, this periodicity of these residues is preferably avoided,especially in human gene therapy applications. The foregoing applies toall protein engineering aspects of the invention, including withoutlimitation the engineering of point mutations into receptor domains, andto the choice or design of boundaries between the various proteindomains.

Other Components, Design Features and Applications

The chimeric proteins may contain as a heterologous domain a cellularlocalization domain such as a membrane retention domain. See e.g.PCT/US94/01617, especially pages 26–27. Briefly, a membrane retentiondomain can be isolated from any convenient membrane-bound protein,whether endogenous to the host cell or not. The membrane retentiondomain may be a transmembrane retention domain, i.e., an amino acidsequence which extends across the membrane as in the case of cellsurface proteins, including many receptors. The transmembrane peptidesequence may be extended to span part or all of an extracellular and/orintracellular domain as well. Alternatively, the membrane retentiondomain may be a lipid membrane retention domain such as a myristoylationor palmitoylation site which permits association with the lipids of thecell surface membrane. Lipid membrane retention domains will usually beadded at the 5′ end of the coding sequence for N-terminal binding to themembrane and, proximal to the 3′ end for C-terminal binding. Peptidesequences involving post-translational processing to provide for lipidmembrane binding are described by Carr, et al., PNAS USA (1988) 79,6128; Aitken, et al., FEBS Lett. (1982) 150, 314; Henderson, et al.,PNAS USA (1983) 80, 319; Schulz, et al., Virology (1984), 123, 2131;Dellman, et al., Nature (1985) 314, 374; and reviewed in Ann. Rev. ofBiochem. (1988) 57, 69. An amino acid sequence of interest includes thesequence M-G-S-S-K-S-K-P-K-D-P-S-Q-R. Various DNA sequences can be usedto encode such sequences in the various chimeric proteins of thisinvention. Other localization domains include organelle-targetingdomains and sequences such as -K-D-E-L and -H-D-E-L which targetproteins bearing them to the endoplasmic reticulum, as well as nuclearlocalization sequences which are particularly useful for chimericproteins designed for (direct) transcriptional regulation. Variouscellular localization sequences and signals are well known in the art.

Further details which may be used in the practice of the subjectinvention relating to the design, assembly and use of constructsencoding chimeric proteins containing various effector domains includingcytoplasmic signal initiation domains such as the CD3 zeta chain,nuclear transcription factor domains including among others VP16 andGAL4, domains capable of triggering apoptosis including the Fascytoplasmic domain and others are disclosed in PCT/US94/01617 andPCT/US95/10591. The latter international application further disclosesadditional features particularly applicable to the creation ofgenetically engineered animals which may be used as disease models inbiopharmaceutical research. Those features include the use of tissuespecific regulatory elements in the constructs for expression of thechimeric proteins and the application of regulated transcription to theexpression of Cre recombinase as the target gene leading to theelimination of a gene of interest flanked by loxP sequences.Alternatively, flp and its cognate recognition sequences may be usedinstead of Cre and lox. Those features may be adapted to the subjectinvention.

In various cases, especially in embodiments involving whole animalscontaining cells engineered in accordance with this invention, it willoften be preferred, and in some cases required, that the various domainsof the chimeric proteins be derived from proteins of the same species asthe host cell. Thus, for genetic engineering of human cells, it is oftenpreferred that the heterologous domains (as well as the FKBP and FRBdomains) be of human origin, rather than of bacterial, yeast or othernon-human source.

Epitope tags may also be incorporated into chimeric proteins of thisinvention to permit convenient detection.

Tissue-Specific or Cell-Type Specific Expression

It will be preferred in certain embodiments, that the chimeric proteinsbe expressed in a cell-specific or tissue-specific manner. Suchspecificity of expression may be achieved by operably linking one oremore of the DNA sequences encoding the chimeric protein(s) to acell-type specific transcriptional regulatory sequence (e.g.promoter/enhancer). Numerous cell-type specific transcriptionalregulatory sequences are known. Others may be obtained from genes whichare expressed in a cell-specific manner. See e.g. PCT/US95/10591,especially pp. 36–37.

For example, constructs for expressing the chimeric proteins may containregulatory sequences derived from known genes for specific expression inselected tissues.

Representative examples are tabulated below:

Tissue Gene Reference lens g2-crystallin Breitman, M. L., Clapoff, S.,Rossant, J., Tsui, L. C., Golde, L. M., Maxwell, I. H., Bernstin, A.(1987) Genetic Ablation: targeted expression of a toxin gene causesmicrophthalmia in transgenic mice. Science 238: 1563–1565 aA-crystallinLandel, C. P., Zhao, J., Bok, D., Evans, G. A. (1988) Lens-specificexpression of a recombinant ricin induces developmental defects in theeyes of transgenic mice. Genes Dev. 2: 1168–1178 Kaur, S., key, B.,Stock, J., McNeish, J. D., Akeson, R., Potter, S. S. (1989) Targetedablation of alpha-crystallin-synthesizing cells produces lens-deficienteyes in transgenic mice. Development 105: 613–619 pituitary - GrowthBehringer, R. R., Mathews, L. S., Palmiter, soma- hormone R. D.,Brinster, R. L. (1988) Dwarf mice trophic produced by genetic ablationof growth cells hormone-expressing cells. Genes Dev. 2: 453–461 pancreasInsulin- Ornitz, D. M., Palmiter, R. D., Hammer, Elastase - R. E.,Brinster, R. L., Swift, G. H., acinar cell MacDonald, R. J. (1985)Specific expression specific of an elastase-human growth fusion inpancreatic acinar cells of transgeneic mice. Nature 131: 600–603Palmiter, R. D., Behringer, R. R., Quaife, C. J., Maxwell, F., Maxwell,I. H., Brinster, R. L. (1987) Cell lineage ablation in transgeneic miceby cell-specific expression of a toxin gene. Cell 50: 435–443 T cells1ck promoter Chaffin, K. E., Beals, C. R., Wilkie, T. M., Forbush, K.A., Simon, M. I., Perlmutter, R. M. (1990) EMBO Journal 9: 3821–3829 Bcells Immuno- Borelli, E., Heyman, R., Hsi, M., Evans, globulin R. M.(1988) Targeting of an inducible toxic kappa light phenotype in animalcells. Proc. Natl. Acad. chain Sci. USA 85: 7572–7576 Heyman, R. A.,Borrelli, E., Lesley, J., Anderson, D., Richmond, D. D., Baird, S. M.,Hyman, R., Evans, R. M. (1989) Thymidine kinase obliteration: creationof transgenic mice with controlled immunodeficiencies. Proc. Natl. Acad.Sci. USA 86: 2698–2702 Schwann P₀ promoter Messing, A., Behringer, R.R., Hammang, cells J. P. Palmiter, R D, Brinster, R L, Lemke, G., P0promoter directs espression of reporter and toxin genes to Schwann cellsof transgenic mice. Neuron 8: 507–520 1992 Myelin basic Miskimins, R.Knapp, L., Dewey, M J, Zhang, protein X. Cell and tissue-specificexpression of a heterologous gene under control of the myelin basicprotein gene promoter in trangenic mice. Brain Res Dev Brain Res 1992Vol 65: 217–21 spermatids protamine Breitman, M. L., Rombola, H.,Maxwell, I. H., Klintworth, G. K., Bernstein, A. (1990) Genetic ablationin transgenic mice with attenuated diphtheria toxin A gene. Mol. Cell.Biol. 10: 474–479 lung Lung Ornitz, D. M., Palmiter, R. D., Hammer,surfactant R. E., Brinster, R. L., Swift, G. H., gene MacDonald, R. J.(1985) Specific expression of an elastase-human growth fusion inpancreatic acinar cells of transgeneic mice. Nature 131: 600–603adipocyte Ross, S. R, Braves, R A, Spiegelman, B M P2 Targetedexpression of a toxin gene to adipose tissue: transgenic mice resistantto obesity Genes and Dev 7: 1318–24 1993 muscle myosin light Lee, K J,Ross, R S, Rockman, H A, Harris, chain A N, O'Brien, T X, van-Bilsen,M., Shubeita, H E, Kandolf, R., Brem, G., Prices et al J. BIol. Chem.1992 Aug. 5, 267: 15875–85 Alpha actin Muscat, G E., Perry, S.,Prentice, H. Kedes, L. The human skeletal alpha-actin gene is regulatedby a muscle-specific enhancer that binds three nuclear factors. GeneExpression 2, 111–26, 1992 . . . / . . . neurons neuro- Reeben, M.Halmekyto, M. Alhonen, L. filament Sinervirta, R. Saarma, M. Janne, J.proteins Tissue-specific expression of rat light neurofilamentpromoter-driven reporter gene in transgenic mice. BBRC 1993: 192: 465–70liver tyrosine amino- transferase, albumin, apolipo- proteinsTarget Gene Constructs

In embodiments of the invention in which the chimeric proteins aredesigned such that their multimerization activates transcription of atarget gene, an appropriate target gene construct is also used in theengineered cells. Appropriate target gene constructs are thosecontaining a target gene and a cognate transcriptional control elementsuch as a promoter and/or enhancer which is responsive to themultimerization of the chimeric proteins. In embodiments involvingdirect activation of transcription, that responsiveness may be achievedby the presence in the target gene construct of one or more DNAsequences recognized by the DNA-binding domain of a chimeric protein ofthis invention (i.e., a DNA sequence to which the chimeric proteinbinds). In embodiments involving indirect activation of transcription,responsiveness may be achieved by the presence in the target geneconstruct of a promoter and/or enhancer sequence which is activated byan intracellular signal generated by multimerization of the chimericproteins. For example, where the chimeric proteins contain the TCR zetachain intracellular domain, the target gene is linked to and under theexpression control of the IL-2 promoter region.

This invention also provides target DNA constructs containing (a) acognate DNA sequence, e.g. to which a DNA-binding chimeric protein ofthis invention is capable of binding (or which is susceptible toindirect activation as discussed above), and (b) flanking DNA sequencefrom the locus of a desired target gene endogenous to the host cells.These constructs permit homologous recombination of the cognate DNAsequence into a host cell in association with an endogenous target gene.In other embodiments the construct contains a desired gene and flankingDNA sequence from a target locus permitting the homologous recombinationof the target gene into the desired locus. Such a target construct mayalso contain the cognate DNA sequence, or the cognate DNA sequence maybe provided by the locus.

The target gene in any of the foregoing embodiments may encode forexample a surface membrane protein (such as a receptor protein), asecreted protein, a cytoplasmic protein, a nuclear protein, arecombinase such as Cre, a ribozyme or an antisense RNA. SeePCT/US94/01617 for general design and construction details and forvarious applications including gene therapy and see PCT/US95/10591regarding applications to animal models of disease.

This invention encompasses a variety of configurations for the chimericproteins. In all cases involving the activation of target genetranscription, however, the chimeric proteins share an importantcharacteristic: cells containing constructs encoding the chimeras and atarget gene construct express the target gene at least one, preferablyat least two, and more preferably at least three or four or more ordersof magnitude more in the presence of the multimerizing ligand than inits absence. Optimally, expression of the selected gene is not observedunless the cells are or have been exposed to a multimerizing ligand.

To recap, the chimeric proteins are capable of initiating a detectablelevel of transcription of target genes within the engineered cells uponexposure of the cells to the a C3 rapalog, i.e., followingmultimerization of the chimeras. Thus, transcription of target genes isactivated in genetically engineered cells of this invention followingexposure of the cells to a C3 rapalog capable of multimerizing thechimeric protein molecules. Said differently, genetically engineeredcells of this invention contain chimeric proteins as described above andare responsive to the presence and/or concentration of a C3 rapalogwhich is capable of multimerizing those chimeric protein molecules. Thatresponsiveness is manifested by the activation of transcription of atarget gene. Such transcriptional activity can be readily detected byany conventional assays for transcription of the target gene. In otherembodiments, the biological response to ligand-mediated multimerizationof the chimeras is cell death or other biological events rather thandirect activation of transcription of a target gene.

Design and Assembly of the DNA Constructs

Constructs may be designed in accordance with the principles,illustrative examples and materials and methods disclosed in the patentdocuments and scientific literature cited herein, each of which isincorporated herein by reference, with modifications and furtherexemplification as described herein. Components of the constructs can beprepared in conventional ways, where the coding sequences and regulatoryregions may be isolated, as appropriate, ligated, cloned in anappropriate cloning host, analyzed by restriction or sequencing, orother convenient means. Particularly, using PCR, individual fragmentsincluding all or portions of a functional unit may be isolated, whereone or more mutations may be introduced using “primer repair”, ligation,in vitro mutagenesis, etc. as appropriate. In the case of DNA constructsencoding fusion proteins, DNA sequences encoding individual domains andsub-domains are joined such that they constitute a single open readingframe encoding a fusion protein capable of being translated in cells orcell lysates into a single polypeptide harboring all component domains.The DNA construct encoding the fusion protein may then be placed into avector that directs the expression of the protein in the appropriatecell type(s). For biochemical analysis of the encoded chimera, it may bedesirable to construct plasmids that direct the expression of theprotein in bacteria or in reticulocyte-lysate systems. For use in theproduction of proteins in mammalian cells, the protein-encoding sequenceis introduced into an expression vector that directs expression in thesecells. Expression vectors suitable for such uses are well known in theart. Various sorts of such vectors are commercially available.

Constructs encoding the chimeric proteins and target genes of thisinvention can be introduced into the cells as one or more DNA moleculesor constructs, in many cases in association with one or more markers toallow for selection of host cells which contain the construct(s). Theconstruct(s) once completed and demonstrated to have the appropriatesequences may then be introduced into a host cell by any convenientmeans. The constructs may be incorporated into vectors capable ofepisomal replication (e.g. BPV or EBV vectors) or into vectors designedfor integration into the host cells' chromosomes. The constructs may beintegrated and packaged into non-replicating, defective viral genomeslike Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus(HSV) or others, including retroviral vectors, for infection ortransduction into cells. Viral delivery systems are discussed in greaterdetail below. Alternatively, the construct may be introduced byprotoplast fusion, electro-poration, biolistics, calcium phosphatetransfection, lipofection, microinjection of DNA or the like. The hostcells will in some cases be grown and expanded in culture beforeintroduction of the construct(s), followed by the appropriate treatmentfor introduction of the construct(s) and integration of theconstruct(s). The cells will then be expanded and screened by virtue ofa marker present in the constructs. Various markers which may be usedsuccessfully include hprt, neomycin resistance, thymidine kinase,hygromycin resistance, etc., and various cell-surface markers such asTac, CD8, CD3, Thyl and the NGF receptor.

In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, one can delete and/or replace anendogenous gene (at the same locus or elsewhere) with a recombinanttarget construct of this invention. For homologous recombination, onemay generally use either ½ or O-vectors. See, for example, Thomas andCapecchi, Cell (1987) 51, 503–512; Mansour, et al., Nature (1988) 336,348–352; and Joyner, et al., Nature (1989) 338, 153–156.

The constructs may be introduced as a single DNA molecule encoding allof the genes, or different DNA molecules having one or more genes. Theconstructs may be introduced simultaneously or consecutively, each withthe same or different markers.

Vectors containing useful elements such as bacterial or yeast origins ofreplication, selectable and/or amplifiable markers, promoter/enhancerelements for expression in procaryotes or eucaryotes, and mammalianexpression control elements, etc. which may be used to prepare stocks ofconstruct DNAs and for carrying out transfections are well known in theart, and many are commercially available.

Delivery of Nucleic Acid: Ex Vivo and In Vivo

Any means for the introduction of heterologous nucleic acids into hostcells, especially eucaryotic cells, an in particular animal cells,preferably human or non-human mammalian cells, may be adapted to thepractice of this invention. For the purpose of this discussion, thevarious nucleic acid constructs described herein may together bereferred to as the transgene. Ex vivo approaches for delivery of DNAinclude calcium phosphate precipitation, electroporation, lipofectionand infection via viral vectors. Two general in vivo gene therapyapproaches include (a) the delivery of “naked”, lipid-complexed orliposome-formulated or otherwise formulated DNA and (b) the delivery ofthe heterologous nucleic acids via viral vectors. In the formerapproach, prior to formulation of DNA, e.g. with lipid, a plasmidcontaining a transgene bearing the desired DNA constructs may first beexperimentally optimized for expression (e.g., inclusion of an intron inthe 5′ untranslated region and elimination of unnecessary sequences(Felgner, et al., Ann NY Acad Sci 126–139, 1995). Formulation of DNA,e.g. with various lipid or liposome materials, may then be effectedusing known methods and materials and delivered to the recipient mammal.

While various viral vectors may be used in the practice of thisinvention, retroviral-, AAV- and adenovirus-based approaches are ofparticular interest. See, for example, Dubensky et al. (1984) Proc.Natl. Acad. Sci. USA 81, 7529–7533; Kaneda et al., (1989) Science243,375–378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86,3594–3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285–17293 andFerry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377–8381. Thefollowing additional guidance on the choice and use of viral vectors maybe helpful to the practitioner.

Retroviral Vectors

Retroviruses are a class of RNA viruses in which the RNA genome isreversely transcribed to DNA in the infected cell. The retroviral genomecan integrate into the host cell genome and requires three viral genes,gag, pol and env, as well as the viral long terminal repeats (LTRs). TheLTRs also act as enhancers and promoters for the viral genes. Thepackaging sequence of the virus, (Y), allows the viral RNA to bedistinguished from other RNAs in the cell (Verma et al., Nature389:239–242, 1997). For expression of a foreign gene, the viral proteinsare replaced with the gene of interest in the viral vector, which isthen transfected into a packaging line containing the viral packagingcomponents. Packaged virus is secreted from the packaging line into theculture medium, which can then be used to infect cells in culture. Sinceretroviruses are unable to infect non-dividing cells, they have beenused primarily for ex vivo gene therapy.

AAV Vectors

Adeno-associated virus (AAV)-based vectors are of general interest as adelivery vehicle to various tissues, including muscle and lung. AAVvectors infect cells and stably integrate into the cellular genome withhigh frequency. AAV can infect and integrate into growth-arrested cells(such as the pulmonary epithelium), and is non-pathogenic.

The AAV-based expression vector to be used typically includes the 145nucleotide AAV inverted terminal repeats (ITRs) flanking a restrictionsite that can be used for subcloning of the transgene, either directlyusing the restriction site available, or by excision of the transgenewith restriction enzymes followed by blunting of the ends, ligation ofappropriate DNA linkers, restriction digestion, and ligation into thesite between the ITRs. The capacity of AAV vectors is about 4.4 kb. Thefollowing proteins have been expressed using various AAV-based vectors,and a variety of promoter/enhancers: neomycin phosphotransferase,chloramphenicol acetyl transferase, Fanconi's anemia gene, cysticfibrosis transmembrane conductance regulator, and granulocyte macrophagecolony-stimulating factor (Kotin, R. M., Human Gene Therapy 5:793–801,1994, Table I). A transgene incorporating the various DNA constructs ofthis invention can similarly be included in an AAV-based vector. As analternative to inclusion of a constitutive promoter such as CMV to driveexpression of the recombinant DNA encoding the fusion protein(s), an AAVpromoter can be used (ITR itself or AAV p5 (Flotte, et al. J. Biol.Chem. 268:3781–3790, 1993)).

Such a vector can be packaged into AAV virions by reported methods. Forexample, a human cell line such as 293 can be co-transfected with theAAV-based expression vector and another plasmid containing open readingframes encoding AAV rep and cap under the control of endogenous AAVpromoters or a heterologous promoter. In the absence of helper virus,the rep proteins Rep68 and Rep78 prevent accumulation of the replicativeform, but upon superinfection with adenovirus or herpes virus, theseproteins permit replication from the ITRs (present only in the constructcontaining the transgene) and expression of the viral capsid proteins.This system results in packaging of the transgene DNA into AAV virions(Carter, B. J., Current Opinion in Biotechnology 3:533–539, 1992; Kotin,R. M, Human Gene Therapy 5:793–801, 1994)). Methods to improve the titerof AAV can also be used to express the transgene in an AAV virion. Suchstrategies include, but are not limited to: stable expression of theITR-flanked transgene in a cell line followed by transfection with asecond plasmid to direct viral packaging; use of a cell line thatexpresses AAV proteins inducibly, such as temperature-sensitiveinducible expression or pharmacologically inducible expression.Additionally, one may increase the efficiency of AAV transduction bytreating the cells with an agent that facilitates the conversion of thesingle stranded form to the double stranded form, as described in Wilsonet al., WO96/39530.

Concentration and purification of the virus can be achieved by reportedmethods such as banding in cesium chloride gradients, as was used forthe initial report of AAV vector expression in vivo (Flotte, et al. J.Biol. Chem. 268:3781–3790, 1993) or chromatographic purification, asdescribed in O'Riordan et al., WO97/08298.

For additional detailed guidance on AAV technology which may be usefulin the practice of the subject invention, including methods andmaterials for the incorporation of a transgene, the propagation andpurification of the recombinant AAV vector containing the transgene, andits use in transfecting cells and mammals, see e.g. Carter et al, U.S.Pat. No. 4,797,368 (10 Jan. 1989); Muzyczka et al, U.S. Pat. No.5,139,941 (18 Aug. 1992); Lebkowski et al, U.S. Pat. No. 5,173,414 (22Dec. 1992); Srivastava, U.S. Pat. No. 5,252,479 (12 Oct. 1993);Lebkowski et al, U.S. Pat. No. 5,354,678 (11 Oct. 1994); Shenk et al,U.S. Pat. No. 5,436,146 (25 Jul. 1995); Chatterjee et al, U.S. Pat. No.5,454,935 (12 Dec. 1995), Carter et al WO 93/24641 (published 9 Dec.1993), and Flotte et al., U.S. Pat. No. 5,658,776 (19 Aug. 1997).

Adenovirus Vectors

Various adenovirus vectors have been shown to be of use in the transferof genes to mammals, including humans. Replication-deficient adenovirusvectors have been used to express marker proteins and CFTR in thepulmonary epithelium. The first generation E1a deleted adenovirusvectors have been improved upon with a second generation that includes atemperature-sensitive E2a viral protein, designed to express less viralprotein and thereby make the virally infected cell less of a target forthe immune system (Goldman et al., Human Gene Therapy 6:839–851, 1995).More recently, a viral vector deleted of all viral open reading frameshas been reported (Fisher et al., Virology 217:11–22, 1996). Moreover,it has been shown that expression of viral IL-10 inhibits the immuneresponse to adenoviral antigen (Qin et al., Human Gene Therapy8:1365–1374, 1997).

DNA sequences of a number of adenovirus types are available fromGenbank. The adenovirus DNA sequences may be obtained from any of the 41human adenovirus types currently identified. Various adenovirus strainsare available from the American Type Culture Collection, Rockville, Md.,or by request from a number of commercial and academic sources. Atransgene as described herein may be incorporated into any adenoviralvector and delivery protocol, by the same methods (restriction digest,linker ligation or filling in of ends, and ligation) used to insert theCFTR or other genes into the vectors. Hybrid Adenovirus-AAV vectorsrepresented by an adenovirus capsid containing selected portions of theadenovirus sequence, 5′ and 3′ AAV ITR sequences flanking the transgeneand other conventional vector regulatory elements may also be used. Seee.g. Wilson et al, International Patent Application Publication No. WO96/13598. For additional detailed guidance on adenovirus and hybridadenovirus-AAV technology which may be useful in the practice of thesubject invention, including methods and materials for the incorporationof a transgene, the propagation and purification of recombinant viruscontaining the transgene, and its use in transfecting cells and mammals,see also Wilson et al, WO 94/28938, WO 96/13597 and WO 96/26285, andreferences cited therein.

Generally the DNA or viral particles are transferred to a biologicallycompatible solution or pharmaceutically acceptable delivery vehicle,such as sterile saline, or other aqueous or non-aqueous isotonic sterileinjection solutions or suspensions, numerous examples of which are wellknown in the art, including Ringer's, phosphate buffered saline, orother similar vehicles.

Preferably, in gene therapy applications, the DNA or recombinant virusis administered in sufficient amounts to transfect cells at a levelproviding therapeutic benefit without undue adverse effects. Optimaldosages of DNA or virus depends on a variety of factors, as discussedelsewhere, and may thus vary somewhat from patient to patient. Again,therapeutically effective doses of viruses are considered to be in therange of about 20 to about 50 ml of saline solution containingconcentrations of from about 1×10⁷ to about 1×10¹⁰ pfu of virus/ml, e.g.from 1×10 ⁸ to 1×10⁹ pfu of virus/ml.

Host Cells

This invention is particularly useful for the engineering of animalcells and in applications involving the use of such engineered animalcells. The animal cells may be insect, worm or mammalian cells. Whilevarious mammalian cells may be used, including, by way of example,equine, bovine, ovine, canine, feline, murine, and non-human primatecells, human cells are of particular interest. Among the variousspecies, various types of cells may be used, such as hematopoietic,neural, glial, mesenchymal, cutaneous, mucosal, stromal, muscle(including smooth muscle cells), spleen, reticulo-endothelial,epithelial, endothelial, hepatic, kidney, gastrointestinal, pulmonary,fibroblast, and other cell types. Of particular interest arehematopoietic cells, which may include any of the nucleated cells whichmay be involved with the erythroid, lymphoid or myelomonocytic lineages,as well as myoblasts and fibroblasts. Also of interest are stem andprogenitor cells, such as hematopoietic, neural, stromal, muscle,hepatic, pulmonary, gastrointestinal and mesenchymal stem cells.

The cells may be autologous cells, syngeneic cells, allogeneic cells andeven in some cases, xenogeneic cells with respect to an intended hostorganism. The cells may be modified by changing the majorhistocompatibility complex (“MHC”) profile, by inactivatingβ2-microglobulin to prevent the formation of functional Class I MHCmolecules, inactivation of Class II molecules, providing for expressionof one or more MHC molecules, enhancing or inactivating cytotoxiccapabilities by enhancing or inhibiting the expression of genesassociated with the cytotoxic activity, or the like.

In some instances specific clones or oligoclonal cells may be ofinterest, where the cells have a particular specificity, such as T cellsand B cells having a specific antigen specificity or homing target sitespecificity.

Introduction of Constructs into Animals

Cells which have been modified ex vivo with the DNA constructs may begrown in culture under selective conditions and cells which are selectedas having the desired construct(s) may then be expanded and furtheranalyzed, using, for example, the polymerase chain reaction fordetermining the presence of the construct in the host cells and/orassays for the production of the desired gene product(s). Once modifiedhost cells have been identified, they may then be used as planned, e.g.grown in culture or introduced into a host organism.

Depending upon the nature of the cells, the cells may be introduced intoa host organism, e.g. a mammal, in a wide variety of ways. Hematopoieticcells may be administered by injection into the vascular system, therebeing usually at least about 10⁴ cells and generally not more than about10¹⁰ cells. The number of cells which are employed will depend upon anumber of circumstances, the purpose for the introduction, the lifetimeof the cells, the protocol to be used, for example, the number ofadministrations, the ability of the cells to multiply, the stability ofthe therapeutic agent, the physiologic need for the therapeutic agent,and the like. Generally, for myoblasts or fibroblasts for example, thenumber of cells will be at least about 104 and not more than about 109and may be applied as a dispersion, generally being injected at or nearthe site of interest. The cells will usually be in aphysiologically-acceptable medium.

Cells engineered in accordance with this invention may also beencapsulated, e.g. using conventional biocompatible materials andmethods, prior to implantation into the host organism or patient for theproduction of a therapeutic protein. See e.g. Hguyen et al, TissueImplant Systems and Methods for Sustaining viable High Cell Densitieswithin a Host, U.S. Pat. No. 5,314,471 (Baxter International, Inc.);Uludag and Sefton, 1993, J. Biomed. Mater. Res. 27 (10):1213–24 (HepG2cells/hydroxyethyl methacrylate-methyl methacrylate membranes); Chang etal, 1993, Hum Gene Ther 4 (4):433–40 (mouse Ltk-cells expressinghGH/immunoprotective perm-selective alginate microcapsules; Reddy et al,1993, J Infect Dis 168 (4):1082–3 (alginate); Tai and Sun, 1993, FASEB J7 (11):1061–9 (mouse fibroblasts expressinghGH/alginate-poly-L-lysine-alginate membrane); Ao et al, 1995,Transplantation Proc. 27 (6):3349, 3350 (alginate); Rajotte et al, 1995,Transplantation Proc. 27 (6):3389 (alginate); Lakey et al, 1995,Transplantation Proc. 27 (6):3266 (alginate); Korbutt et al, 1995,Transplantation Proc. 27 (6):3212 (alginate); Dorian et al, U.S. Pat.No. 5,429,821 (alginate); Emerich et al, 1993, Exp Neurol 122 (1):37–47(polymer-encapsulated PC12 cells); Sagen et al, 1993, J Neurosci 13(6):2415–23 (bovine chromaffin cells encapsulated in semipermeablepolymer membrane and implanted into rat spinal subarachnoid space);Aebischer et al, 1994, Exp Neurol 126 (2):151–8 (polymer-encapsulatedrat PC12 cells implanted into monkeys; see also Aebischer, WO 92/19595);Savelkoul et al, 1994, J Immunol Methods 170 (2):185–96 (encapsulatedhybridomas producing antibodies; encapsulated transfected cell linesexpressing various cytokines); Winn et al, 1994, PNAS USA 91 (6):2324–8(engineered BHK cells expressing human nerve growth factor encapsulatedin an immunoisolation polymeric device and transplanted into rats);Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry 18(5):935–46 (polymer-encapsulated PC12 cells implanted into rats);Kordower et al, 1994, PNAS USA 91 (23):10898–902 (polymer-encapsulatedengineered BHK cells expressing hNGF implanted into monkeys) and Butleret al WO 95/04521 (encapsulated device). The cells may then beintroduced in encapsulated form into an animal host, preferably a mammaland more preferably a human subject in need thereof. Preferably theencapsulating material is semipermeable, permitting release into thehost of secreted proteins produced by the encapsulated cells. In manyembodiments the semipermeable encapsulation renders the encapsulatedcells immunologically isolated from the host organism in which theencapsulated cells are introduced. In those embodiments the cells to beencapsulated may express one or more chimeric proteins containingcomponent domains derived from proteins of the host species and/or fromviral proteins or proteins from species other than the host species. Forexample in such cases the chimeras may contain elements derived fromGAL4 and VP16. The cells may be derived from one or more individualsother than the recipient and may be derived from a species other thanthat of the recipient organism or patient.

Instead of ex vivo modification of the cells, in many situations one maywish to modify cells in vivo. For this purpose, various techniques havebeen developed for modification of target tissue and cells in vivo. Anumber of viral vectors have been developed, such as adenovirus,adeno-associated virus, and retroviruses, as discussed above, whichallow for transfection and, in some cases, integration of the virus intothe host. See, for example, Dubensky et al. (1984) Proc. Natl. Acad.Sci. USA 81, 7529–7533; Kaneda et al., (1989) Science 243,375–378;Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3594–3598; Hatzogluet al. (1990) J. Biol. Chem. 265, 17285–17293 and Ferry, et al. (1991)Proc. Natl. Acad. Sci. USA 88, 8377–8381. The vector may be administeredby injection, e.g. intravascularly or intramuscularly, inhalation, orother parenteral mode. Non-viral delivery methods such as administrationof the DNA via complexes with liposomes or by injection, catheter orbiolistics may also be used.

In accordance with in vivo genetic modification, the manner of themodification will depend on the nature of the tissue, the efficiency ofcellular modification required, the number of opportunities to modifythe particular cells, the accessibility of the tissue to the DNAcomposition to be introduced, and the like. By employing an attenuatedor modified retrovirus carrying a target transcriptional initiationregion, if desired, one can activate the virus using one of the subjecttranscription factor constructs, so that the virus may be produced andtransfect adjacent cells.

The DNA introduction need not result in integration in every case. Insome situations, transient maintenance of the DNA introduced may besufficient. In this way, one could have a short term effect, where cellscould be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

Binding properties, Assays

Rapamycin is known to bind to the human protein, FKBP12 and to form atripartite complex with hFKBP12 and FRAP, a human counterpart to theyeast proteins TOR1 and TOR2. Rapalogs may be characterized and comparedto rapamycin with respect to their ability to bind to human FKBP12and/or to form tripartite complexes with human FKBP12 and human FRAP (orfusion proteins or fragments containing its FRB domain). See WO 96/41865(Clackson et al). That application discloses various materials andmethods which can be used to quantify the ability of a compound to bindto human FKBP12 or to form a tripartite complex with (i.e.,“heterodimerize”) proteins comprising human FKBP12 and the FRB domain ofhuman FRAP, respectively. Such assays include fluorescence polarizationassays to measure binding. Also included are cell based transcriptionassays in which the ability of a compound to form the tripartite complexis measured indirectly by correlation with the observed level ofreporter gene product produced by engineered mammalian cells in thepresence of the compound. Corresponding cell-based assays may also beconducted in engineered yeast cells. See e.g. WO 95/33052 (Berlin etal).

It will often be preferred that the rapalogs of this invention bephysiologically acceptable (i.e., lack undue toxicity toward the cell ororganism with which it is to be used), can be taken orally by animals(i.e., is orally active in applications in whole animals, including genetherapy), and/or can cross cellular and other membranes, as necessaryfor a particular application.

In addition, preferred rapalogs are those which bind preferentially tomutant immunophilins (by way of non-limiting example, a human FKBP inwhich Phe36 is replaced with a different amino acid, preferably an aminoacid with a less bulky R group such as valine or alanine) over native ornaturally-occurring immunophilins. For example, such compounds may bindpreferentially to mutant FKBPs at least an order of magnitude betterthan they bind to human FKBP12, and in some cases may bind to mutantFKBPs greater than 2 or even 3 or more orders of magnitude better thanthey do to human FKBP12, as determined by any scientifically valid orart-accepted assay methodology.

Binding affinities of various rapalogs of this invention with respect tohuman FKBP12, variants thereof or other immunophilin proteins may bedetermined by adaptation of known methods used in the case of FKBP. Forinstance, the practitioner may measure the ability of a compound of thisinvention to compete with the binding of a known ligand to the proteinof interest. See e.g. Sierkierka et al, 1989, Nature 341, 755–757 (testcompound competes with binding of labeled FK506 derivative to FKBP).

One set of preferred rapalogs of this invention which binds, to humanFKBP12, to a mutant thereof as discussed above, or to a fusion proteincontaining such FKBP domains, with a Kd value below about 200 nM, morepreferably below about 50 nM, even more preferably below about 10 nM,and even more preferably below about 1 nM, as measured by direct bindingmeasurement (e.g. fluorescence quenching), competition bindingmeasurement (e.g. versus FK506), inhibition of FKBP enzyme activity(rotamase), or other assay methodology. In one subset of such compounds,the FKBP domain is one in which phenylalanine at position 36 has beenreplaced with an amino acid having a less bulky side chain, e.g.alanine, valine, methionine or serine.

A Competitive Binding FP Assay is described in detail in WO96/41865.That assay permits the in vitro measurement of an IC50 value for a givencompound which reflects its ability to bind to an FKBP protein incompetition with a labeled FKBP ligand, such as, for example, FK506.

One preferred class of compounds of this invention are those rapalogswhich have an IC50 value in the Competitive Binding FP Assay better than1000 nM, preferably better than 300 nM, more preferably better than 100nM, and even more preferably better than 10 nM with respect to a givenFKBP domain and ligand pair, e.g. human FKBP12 or a variant thereof withup to 10, preferably up to 5 amino acid replacements, with aflouresceinated FK506 standard. In one subset of that class, the FKBPdomain has one of the abovementioned modifications at position 36.

The ability of the rapalogs to multimerize chimeric proteins may bemeasured in cell-based assays by measuring the occurrence of an eventtriggered by such multimerization. For instance, one may use cellscontaining and capable of expressing DNA encoding a first chimericprotein comprising one or more FKBP-domains and one or more effectordomains as well as DNA encoding a second chimeric protein containing anFRB domain and one or more effector domains capable, uponmultimerization, of actuating a biological response. We prefer to usecells which further contain a reporter gene under the transcriptionalcontrol of a regulatory element (i.e., promoter) which is responsive tothe multimerization of the chimeric proteins. The design and preparationof illustrative components and their use in so engineered cells isdescribed in WO96/41865 and the other international patent applicationsreferred to in this and the foregoing section. The cells are grown ormaintained in culture. A rapalog is added to the culture medium andafter a suitable incubation period (to permit gene expression andsecretion, e.g. several hours or overnight) the presence of the reportergene product is measured. Positive results, i.e., multimerization,correlates with transcription of the reporter gene as observed by theappearance of the reporter gene product. The reporter gene product maybe a conveniently detectable protein (e.g. by ELISA) or may catalyze theproduction of a conveniently detectable product (e.g. colored).Materials and methods for producing appropriate cell lines forconducting such assays are disclosed in the international patentapplications cited above in this section. Typically used target genesinclude by way of example SEAP, hGH, beta-galactosidase, GreenFluorescent Protein and luciferase, for which convenient assays arecommercially available.

Another preferred class of compounds of this invention are those whichare capable of inducing a detectable signal in a 2-hybrid transcriptionassay based on fusion proteins containing an FKBP domain. Preferably,the FKBP domain is an FKBP domain other than wild-type human FKBP12.

Another assay for measuring the ability of the rapalogs to multimerizechimeric proteins, like the FKBP-based transcription assay, is acell-based assay which measures the occurrence of an event triggered bysuch multimerization. In this case, one uses cells which constitutivelyexpress a detectable product. The cells also contain and are capable ofexpressing DNAs encoding chimeric proteins comprising one or moreimmunophilin-derived ligand binding domains and one or more effectordomains, such as the intracellular domain of FAS, capable, uponmultimerization, of triggering cell death. The design and preparation ofillustrative components and their use in so engineering cells isdescribed in WO95/02684. See also WO96/41865. The cells are maintainedor cultured in a culture medium permitting cell growth or continuedviability. The cells or medium are assayed for the presence of theconstitutive cellular product, and a base-line level of reporter is thusestablished. One may use cells engineered for constitutive production ofhGH or any other conveniently detectable product to serve as thereporter. The compound to be tested is added to the medium, the cellsare incubated, and the cell lysate or medium is tested for the presenceof reporter at one or more time points. Decrease in reporter productionindicates cell death, an indirect measure of multimerization of thefusion proteins.

Another preferred class of compounds of this invention are those whichare capable of inducing a detectable signal in such an FKBP/FRB-basedapoptosis assay. Preferably, the FKBP domain is an FKBP domain otherthan wild-type human FKBP12. In some cases, the FKBP domain is modified,as discussed above. Also preferably, the FRB domain is an FRB domainother than wild-type FRB from human FRAP. In some cases, the FRB domainis modified at position 2098, as described above.

Conducting such assays permits the practitioner to select rapalogspossessing the desired IC50 values and/or binding preference for amutant FKBP over wild-type human FKBP12. The Competitive Binding FPAssay permits one to select monomers or rapalogs which possess thedesired IC50 values and/or binding preference for a mutant FKBP orwild-type FKBP relative to a control, such as FK506.

Applications

The rapalogs can be used as described in WO94/18317, WO95/02684,WO96/20951, WO95/41865, e.g. to regulatably activate the transcriptionof a desired gene, delete a target gene, actuate apoptosis, or triggerother biological events in engineered cells growing in culture or inwhole organisms, including in gene therapy applications. The followingare non-limiting examples of applications of the subject invention.

1. Regulated gene therapy. In many instances, the ability to switch atherapeutic gene on and off at will or the ability to titrate expressionwith precision are important for therapeutic efficacy. This invention isparticularly well suited for achieving regulated expression of atherapeutic target gene in the context of human gene therapy. Oneexample uses a pair of chimeric proteins (one containing at least oneFRB domain, the other containing at least one FKBP domain), a C3 rapalogof this invention capable of dimerizing the chimeras, and a target geneconstruct to be expressed. One of the chimeric proteins comprises aDNA-binding domain, preferably a composite DNA-binding domain asdescribed in Pomerantz et al, supra, as the heterologous effectordomain. The second chimeric protein comprises a transcriptionalactivating domain as the heterologous effector domain. The C3 rapalog iscapable of binding to both chimeras and thus of effectivelycross-linking the chimeras. DNA molecules encoding and capable ofdirecting the expression of these chimeric proteins are introduced intothe cells to be engineered. Also introduced into the cells is a targetgene linked to a DNA sequence to which the DNA-binding domain is capableof binding. Contacting the engineered cells or their progeny with the C3rapalog (by administering it to the animal or patient) leads to assemblyof the transcription factor complex and hence to expression of thetarget gene. The design and use of similar components is disclosed inPCT/US93/01617 and in WO 96/41865 (Clackson et al). In practice, thelevel of target gene expression should be a function of the number orconcentration of chimeric transcription factor complexes, which shouldin turn be a function of the concentration of the C3 rapalog. Dose (ofC3 rapalog)-responsive gene expression is typically observed.

The C3 rapalog may be administered to the patient as desired to activatetranscription of the target gene. Depending upon the binding affinity ofthe C3 rapalog, the response desired, the manner of administration, thebiological half-life of the rapalog and/or target gene mRNA, the numberof engineered cells present, various protocols may be employed. The C3rapalog may be administered by various routes, including parenterally ororally. The number of administrations will depend upon the factorsdescribed above. The C3 rapalog may be taken orally as a pill, powder,or dispersion; bucally; sublingually; injected intravascularly,intraperitoneally, intramuscularly, subcutaneously; by inhalation, orthe like. The C3 rapalog (and monomeric antagonist compound) may beformulated using conventional methods and materials well known in theart for the various routes of administration. The precise dose andparticular method of administration will depend upon the above factorsand be determined by the attending physician or human or animalhealthcare provider. For the most part, the manner of administrationwill be determined empirically.

In the event that transcriptional activation by the C3 rapalog is to bereversed or terminated, a monomeric compound which can compete with theC3 rapalog may be administered. Thus, in the case of an adverse reactionor the desire to terminate the therapeutic effect, an antagonist to thedimerizing agent can be administered in any convenient way, particularlyintravascularly, if a rapid reversal is desired. Alternatively, one mayprovide for the presence of an inactivation domain (or transcriptionalsilencer) with a ligand binding domain. In another approach, cells maybe eliminated through apoptosis via signalling through Fas or TNFreceptor as described elsewhere. See International Patent ApplicationsPCT/US94/01617 and PCT/US94/08008.

The particular dosage of the C3 rapalog for any application may bedetermined in accordance with the procedures used for therapeutic dosagemonitoring, where maintenance of a particular level of expression isdesired over an extended period of times, for example, greater thanabout two weeks, or where there is repetitive therapy, with individualor repeated doses of C3 rapalog over short periods of time, withextended intervals, for example, two weeks or more. A dose of the C3rapalog within a predetermined range would be given and monitored forresponse, so as to obtain a time-expression level relationship, as wellas observing therapeutic response. Depending on the levels observedduring the time period and the therapeutic response, one could provide alarger or smaller dose the next time, following the response. Thisprocess would be iteratively repeated until one obtained a dosage withinthe therapeutic range. Where the C3 rapalog is chronically administered,once the maintenance dosage of the C3 rapalog is determined, one couldthen do assays at extended intervals to be assured that the cellularsystem is providing the appropriate response and level of the expressionproduct.

It should be appreciated that the system is subject to many variables,such as the cellular response to the C3 rapalog, the efficiency ofexpression and, as appropriate, the level of secretion, the activity ofthe expression product, the particular need of the patient, which mayvary with time and circumstances, the rate of loss of the cellularactivity as a result of loss of cells or expression activity ofindividual cells, and the like.

2. Production of recombinant proteins and viruses. Production ofrecombinant therapeutic proteins for commercial and investigationalpurposes is often achieved through the use of mammalian cell linesengineered to express the protein at high level. The use of mammaliancells, rather than bacteria or yeast, is indicated where the properfunction of the protein requires post-translational modifications notgenerally performed by heterologous cells. Examples of proteins producedcommercially this way include erythropoietin, tissue plasminogenactivator, clotting factors such as Factor VIII:c, antibodies, etc. Thecost of producing proteins in this fashion is directly related to thelevel of expression achieved in the engineered cells. A secondlimitation on the production of such proteins is toxicity to the hostcell: Protein expression may prevent cells from growing to high density,sharply reducing production levels. Therefore, the ability to tightlycontrol protein expression, as described for regulated gene therapy,permits cells to be grown to high density in the absence of proteinproduction. Only after an optimum cell density is reached, is expressionof the gene activated and the protein product subsequently harvested.

A similar problem is encountered in the construction and use of“packaging lines” for the production of recombinant viruses forcommercial (e.g., gene therapy) and experimental use. These cell linesare engineered to produce viral proteins required for the assembly ofinfectious viral particles harboring defective recombinant genomes.Viral vectors that are dependent on such packaging lines includeretrovirus, adenovirus, and adeno-associated virus. In the latter case,the titer of the virus stock obtained from a packaging line is directlyrelated to the level of production of the viral rep and core proteins.But these proteins are highly toxic to the host cells. Therefore, it hasproven difficult to generate high-titer recombinant AAV viruses. Thisinvention provides a solution to this problem, by allowing theconstruction of packaging lines in which the rep and core genes areplaced under the control of regulatable transcription factors of thedesign described here. The packaging cell line can be grown to highdensity, infected with helper virus, and transfected with therecombinant viral genome. Then, expression of the viral proteins encodedby the packaging cells is induced by the addition of dimerizing agent toallow the production of virus at high titer.

3. Biological research. This invention is applicable to a wide range ofbiological experiments in which precise control over a target gene isdesired. These include: (1) expression of a protein or RNA of interestfor biochemical purification; (2) regulated expression of a protein orRNA of interest in tissue culture cells (or in vivo, via engineeredcells) for the purposes of evaluating its biological function; (3)regulated expression of a protein or RNA of interest in transgenicanimals for the purposes of evaluating its biological function; (4)regulating the expression of a gene encoding another regulatory protein,ribozyme or antisense molecule that acts on an endogenous gene for thepurposes of evaluating the biological function of that gene. Transgenicanimal models and other applications in which the components of thisinvention may be adapted include those disclosed in PCT/US95/10591.

This invention further provides kits useful for the foregoingapplications. Such kits contain DNA constructs encoding and capable ofdirecting the expression of chimeric proteins of this invention (and maycontain additional domains as discussed above) and, in embodimentsinvolving regulated gene transcription, a target gene constructcontaining a target gene linked to one or more transcriptional controlelements which are activated by the multimerization of the chimericproteins. Alternatively, the target gene construct may contain a cloningsite for insertion of a desired target gene by the practitioner. Suchkits may also contain a sample of a dimerizing agent capable ofdimerizing the two recombinant proteins and activating transcription ofthe target gene.

Formulations, Dosage and Administration

By virtue of its capacity to promote protein—protein interactions, arapalog of this invention may be used in pharmaceutical compositions andmethods for promoting formation of complexes of chimeric proteins ofthis invention in a human or non-human mammal containing geneticallyengineered cells of this invention.

The preferred method of such treatment or prevention is by administeringto the mammal an effective amount of the compound to promote measurableformation of such complexes in the engineered cells, or preferably, topromote measurable actuation of the desired biological event triggeredby such complexation, e.g. transcription of a target gene, apoptosis ofengineered cells, etc.

Therapeutic/Prophylactic Administration & Pharmaceutical Compositions

The rapalogs can exist in free form or, where appropriate, in salt form.Pharmaceutically acceptable salts of many types of compounds and theirpreparation are well-known to those of skill in the art. Thepharmaceutically acceptable salts of compounds of this invention includethe conventional non-toxic salts or the quaternary ammonium salts ofsuch compounds which are formed, for example, from inorganic or organicacids of bases.

The compounds of the invention may form hydrates or solvates. It isknown to those of skill in the art that charged compounds form hydratedspecies when lyophilized with water, or form solvated species whenconcentrated in a solution with an appropriate organic solvent.

This invention also relates to pharmaceutical compositions comprising atherapeutically (or prophylactically) effective amount of the compound,and one or more pharmaceutically acceptable carriers and/or otherexcipients. Carriers include e.g. saline, buffered saline, dextrose,water, glycerol, ethanol, and combinations thereof, and are discussed ingreater detail below. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.The composition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Formulation may involve mixing, granulating and compressing ordissolving the ingredients as appropriate to the desired preparation.

The pharmaceutical carrier employed may be, for example, either a solidor liquid.

Illustrative solid carrier include lactose, terra alba, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and thelike. A solid carrier can include one or more substances which may alsoact as flavoring agents, lubricants, solubilizers, suspending agents,fillers, glidants, compression aids, binders or tablet-disintegratingagents; it can also be an encapsulating material. In powders, thecarrier is a finely divided solid which is in admixture with the finelydivided active ingredient. In tablets, the active ingredient is mixedwith a carrier having the necessary compression properties in suitableproportions, and compacted in the shape and size desired. The powdersand tablets preferably contain up to 99% of the active ingredient.Suitable solid carriers include, for example, calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Illustrative liquid carriers include syrup, peanut oil, olive oil,water, etc. Liquid carriers are used in preparing solutions,suspensions, emulsions, syrups, elixirs and pressurized compositions.The active ingredient can be dissolved or suspended in apharmaceutically acceptable liquid carrier such as water, an organicsolvent, a mixture of both or pharmaceutically acceptable oils or fats.The liquid carrier can contain other suitable pharmaceutical additivessuch as solubilizers, emulsifiers, buffers, preservatives, sweeteners,flavoring agents, suspending agents, thickening agents, colors,viscosity regulators, stabilizers or osmo-regulators. Suitable examplesof liquid carriers for oral and parenteral administration include water(partially containing additives as above, e.g. cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g. glycols) and theirderivatives, and oils (e.g. fractionated coconut oil and arachis oil).For parenteral administration, the carrier can also be an oily estersuch as ethyl oleate and isopropyl myristate. Sterile liquid carriersare useful in sterile liquid form compositions for parenteraladministration. The liquid carrier for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

The carrier or excipient may include time delay material well known tothe art, such as glyceryl monostearate or glyceryl distearate along orwith a wax, ethylcellulose, hydroxypropylmethylcellulose,methylmethacrylate and the like. When formulated for oraladministration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentratewith 1,2-propylene glycol, A. Nattermann & Cie. GmbH) has beenrecognized as providing an acceptable oral formulation for othercompounds, and may be adapted to formulations for various compounds ofthis invention.

A wide variety of pharmaceutical forms can be employed. If a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 g. If a liquid carrier is used, thepreparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable solution or suspension in an ampule or vialor nonaqueous liquid suspension.

To obtain a stable water soluble dosage form, a pharmaceuticallyacceptable salt of the multimerizer may be dissolved in an aqueoussolution of an organic or inorganic acid, such as a 0.3M solution ofsuccinic acid or citric acid. Alternatively, acidic derivatives can bedissolved in suitable basic solutions. If a soluble salt form is notavailable, the compound is dissolved in a suitable cosolvent orcombinations thereof. Examples of such suitable cosolvents include, butare not limited to, alcohol, propylene glycol, polyethylene glycol 300,polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty alcoholsor glycerin hydroxy fatty acids esters and the like in concentrationsranging from 0–60% of the total volume.

Various delivery systems are known and can be used to administer themultimerizer, or the various formulations thereof, including tablets,capsules, injectable solutions, encapsulation in liposomes,microparticles, microcapsules, etc. Methods of introduction include butare not limited to dermal, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and(as is usually preferred) oral routes. The compound may be administeredby any convenient or otherwise appropriate route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. For treatment orprophylaxis of nasal, bronchial or pulmonary conditions, preferredroutes of administration are oral, nasal or via a bronchial aerosol ornebulizer.

In certain embodiments, it may be desirable to administer the compoundlocally to an area in need of treatment; this may be achieved by, forexample, and not by way of limitation, local infusion during surgery,topical application, by injection, by means of a catheter, by means of asuppository, or by means of a skin patch or implant, said implant beingof a porous, non-porous, or gelatinous material, including membranes,such as sialastic membranes, or fibers.

In a specific embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic to ease pain at the side of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a lyophilized powder orwater free concentrate in a hermetically sealed container such as anampoule or sachette indicating the quantity of active agent. Where thecomposition is to be administered by infusion, it can be dispensed withan infusion bottle containing sterile pharmaceutical grade water orsaline. Where the composition is administered by injection, an ampouleof sterile water for injection or saline can be provided so that theingredients may be mixed prior to administration.

Administration to an individual of an effective amount of the compoundcan also be accomplished topically by administering the compound(s)directly to the affected area of the skin of the individual. For thispurpose, the compound is administered or applied in a compositionincluding a pharmacologically acceptable topical carrier, such as a gel,an ointment, a lotion, or a cream, which includes, without limitation,such carriers as water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils.

Other topical carriers include liquid petroleum, isopropyl palmitate,polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) inwater, or sodium lauryl sulfate (5%) in water. Other materials such asanti-oxidants, humectants, viscosity stabilizers, and similar agents maybe added as necessary. Percutaneous penetration enhancers such as Azonemay also be included.

In addition, in certain instances, it is expected that the compound maybe disposed within devices placed upon, in, or under the skin. Suchdevices include patches, implants, and injections which release thecompound into the skin, by either passive or active release mechanisms.

Materials and methods for producing the various formulations are wellknown in the art and may be adapted for practicing the subjectinvention. See e.g. U.S. Pat. Nos. 5,182,293 and 4,837,311 (tablets,capsules and other oral formulations as well as intravenousformulations) and European Patent Application Publication Nos. 0 649 659(published Apr. 26, 1995; illustrative formulation for IVadministration) and 0 648 494 (published Apr. 19, 1995; illustrativeformulation for oral administration).

The effective dose of the compound will typically be in the range ofabout 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg ofmammalian body weight, administered in single or multiple doses.Generally, the compound may be administered to patients in need of suchtreatment in a daily dose range of about 1 to about 2000 mg per patient.

The amount of compound which will be effective in the treatment orprevention of a particular disorder or condition will depend in part onthe characteristics of the fusion proteins to be multimerized, thecharacteristics and location of the genetically engineered cells, and onthe nature of the disorder or condition, which can be determined bystandard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. The precise dosage levelshould be determined by the attending physician or other health careprovider and will depend upon well known factors, including route ofadministration, and the age, body weight, sex and general health of theindividual; the nature, severity and clinical stage of the disease; theuse (or not) of concomitant therapies; and the nature and extent ofgenetic engineering of cells in the patient.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers containing one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical or biological products, which notice reflects approval bythe agency of manufacture, use or sale for human administration. Thenotice or package insert may contain instructions for use of a C3rapalog of this invention, consistent with the disclosure herein.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications and published patents, citedthroughout this application are hereby expressly incorporated byreference.

EXPERIMENTAL EXAMPLES

Synthesis of C3-methallyl-rapamycin and C3-allyl-rapamycin

The synthetic procedure we currently use is a modified version of theprocedure described in Liberles et al., July 1997, Proc. Natl. Acad.Sci. USA 94:7825–7830 for the preparation of C7 rapalogs. (Note that C7and C3 as referred to herein refer to the ring positions C16 and C20,respectively, in the nomenclature of Liberles et al). Twenty two mgs ofrapamycin are placed in a 3 ml flame-dried Wheaton vial equipped with astir bar. The rapamycin is dissolved in 200 μl of methylene chloride andcooled to −40 degrees. Lower temperatures have been found to not work asefficiently. 50 μl of methallyl (or allyl) trimethyl silane (12 equiv.)is added, followed by 40 μl of neat BF3 etherate (13 equivalents). After2 hours the reaction is over, as determined by TLC, and is quenched byaddition of saturated NaHCO₃. The reaction mixture is then washed withbrine and dried with sodium sulfate. (The aqueous washes can beback-extracted with methylene chloride and combined with the reactionmixture.) The samples are filtered with a 0.45 micron Nylon filter andthe solvent is evaporated under vacuum.

The crude product is dissolved in 100 μl of chloroform for injection onthe JAI recycling HPLC described below. Three predominant peaks areobtained that can be separated with baseline resolution afterapproximately 15–20 cycles. (Under certain reaction conditions, a fourthpeak appears which elutes before the other three.) Of the threepredominant peaks, the first is C7-S methallyl rapamycin, as determinedby 1H-NMR. The middle peak is the compound of interest, C3-methallylrapamycin, and the fourth peak is composed of side products resultingfrom oversilylation of rapamycin. Following this procedure, we obtained4.2 mgs of pure C3-methallyl rapamycin. Since unmodified rapamycincoincidentally comigrates precisely with C3-methallyl rapamycin, andcannot be removed efficiently with the JAI, it is critical thatrapamycin be completely consumed during the reaction. Overadditionproducts are far more easily removed than the unreacted startingmaterial.

The JAI-purified methallyl rapamycin can be verified by UV, NMR, andmass spectroscopy and biological activity. The presence of contaminants,such as rapamycin, can be easily determined by UV, as described below.

This same procedure has been used to synthesize C3 allyl-rapamycin, andshould be easily extended to synthesize a variety of other C3derivatives.

Analytical Considerations

One particularly convenient diagnostic is UV spectroscopy. Consistentwith disappearance of the triene, the UV spectra of the C3 compoundshave a maximum absorbance at lambda 238 rather than 274–282, typicallyseen for the C7 (R) or (S) compounds. Since the C3 compounds havebaseline absorbance in the region where rapamycin absorbs best, UVanalysis is a good indicator of sample purity and the presence of anytoxic contaminants.

The loss of the triene in C3-methallyl rapamycin is also reflected inthe 1H NMR. A 1H-NMR of methallyl rapamycin, taken in CDCL3, reveals adiagnostic peak from C5 at 6.0 ppm. The remainder of the olefinicprotons all lie upfield between 5 and 5.6 ppm.

Chromatographic Recovery of the C3 Rapalogs

An instrument made by the Japan Analytical Industry Co., LTD. (JAI)efficiently purifies the C3 rapalogs. The instrument we used was a“LC-908 Recycling Preparative HPLC” and the column we used was “JAIGELGS-310” with dimensions of “20×500 mm”.

Functional Characteristics of C3 Rapalogs

The toxicity of the lead compounds was tested by their ability toinhibit the proliferation of CTLL-2 cells, which are IL-2 dependent andhighly sensitive to rapamycin (IC50<1 nM). Incubation of CTLL-2 cellswith 1 μM C3-methallyl rapamycin had no detectable effect onproliferation.

Likewise, C3-allyl rapamycin and C3-methallyl rapamycin were impaired intheir ability to activate transcription in Jurkat cells transfected withGal4-FKBP3, wild type FRB-VP16, and UAS-SEAP. (These constructs aredescribed in Liberles et al, above, the full contents of which areincorporated herein by reference.) In experiments in which FRB*-VP16(FRB harboring T2098L, W2101F, and K2095P) replaces wild type FRB-VP16,reporter gene activity can be detected at an EC50 near 10 nMC3-methallyl rapamycin or C3-allyl rapamycin; as well, the amplitude ofreporter gene activity is higher than that elicited by therapamycin/wild type FRB-VP16 system. C3 derivatives of rapamycin haveimpaired toxicity (immunosuppressive activity) and can be usedefficiently as dimerizers in conjunction with engineered FRB domains.Preparation of 24(S),30(S)-tetrahydro-C3-rapalogs

24,30-tetrahydro rapamycin is prepared as described elsewhere and isconverted into the desired C3-substituted rapalog by the methoddescribed above.Preparation of 13-fluoro-C3-rapalogs

The title compounds are prepared as described above, substituting13-Fluoro rapamycin for rapamycin.Preparation of 28-fluoro-C3-rapalogs

The title compounds are prepared as described above, substituting28-Fluoro rapamycin for rapamycin.Preparation of 43-epi-C3-rapalogs

The title compounds are prepared as described above, substituting 43-epirapamycin for rapamycin.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A compound selected from the group consisting of one of the followingstructures:

wherein RC3 is an allyl or methallyl moiety, optionally substituted withone or more groups selected from —OH, —SH, —CHO, ═O, —COOH, oxime, —NH₂,halo, trihaloalkyl, cyano, —SO₂CF₃, —OSO₂F, —OS(O)₂R¹¹, —SO₂—NHR¹¹,—NHSO₂R¹¹, aryl or heteroaryl; R¹¹ is H or an aliphatic,heteroaliphatic, aryl or heteroaryl moiety; wherein each aryl moiety isa substituted or unsubstituted phenyl ring, and each heteroaryl moietyis selected from a five- or six-membered heteroaryl ring, and each arylor heteroaryl moiety is optionally substituted with one to fivesubstituents selected from the group consisting of —OH, —SH, —CHO, ═O,—COOH, oxime, —NH₂, halo, trihaloalkyl, cyano, —SO₂—CF₃, —OSO₂F,—OS(O)₂R¹¹, —SO₂—NHR¹¹, —NHSO₂R¹¹, aryl, heteroaryl, C1–C8 alkoxy, C1–C8 branched or straight-chain alkyl, acyl, acyloxy, nitro,dialkoxyphenyl, trisubstituted phenyl, —O-aliphatic-COOH, and—O-aliphatic-NH₂, each aliphatic moiety is selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, andcycloalkynyl moieties, optionally substituted with one or moresubstituents selected from the group consisting of —OH, —SH, —CHO, ═O,—COOH, oxime, —NH₂, halo, trihaloalkyl, cyano, —SO₂—CF₃, —OSO₂F,OS(O)₂R¹¹, —SO₂—NHR¹¹, —NHSO₂R¹¹, aryl and heteroaryl moieties, and eachheteroaliphatic moiety is an aliphatic moiety that contains one or moreoxygen, sulfur, nitrogen, phosphorous or silicon atoms.
 2. The compoundof claim 1, wherein each optionally substituted aryl moiety is selectedfrom the group consisting of phenyl, halophenyl, alkoxyphenyl,dialkoxyphenyl, trialkoxyphenyl, and alkylenedioxyphenyl,; and eachoptionally substituted heteroaryl moiety is selected from the groupconsisting of thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl,isothiazolyl, furazanyl, isoxazolyl, thiazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, and triazinyll moieties.
 3. A compositioncomprising a compound of claim 1 or 2 and one or more pharmaceuticallyacceptable excipients.
 4. A compound of claim 1 or 2, wherein thecompound is at least 95% free from other materials.
 5. A compound ofclaim 1 or 2, wherein the compound is at least 98% free from othermaterials.